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  • 1.
    Aldaeus, Fredrik
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Lin, Yuan
    KTH, School of Engineering Sciences (SCI), Mechanics.
    Amberg, Gustav
    KTH, School of Engineering Sciences (SCI), Mechanics.
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Multi-step dielectrophoresis for separation of particles2006In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1131, no 1-2, p. 261-266Article in journal (Refereed)
    Abstract [en]

    A new concept for separation of particles based on repetitive dielectrophoretic trapping and release in a flow system is proposed. Calculations using the finite element method have been performed to envision the particle behavior and the separation effectiveness of the proposed method. As a model system, polystyrene beads in deionized water and a micro-flow channel with arrays of interdigited electrodes have been used. Results show that the resolution increases as a direct function of the number of trap-and-release steps, and that a difference in size will have a larger influence on the separation than a difference in other dielectrophoretic properties. About 200 trap-and-release steps would be required to separate particles with a size difference of 0.2%. The enhanced separation power of dielectrophoresis with multiple steps could be of great importance, not only for fractionation of particles with small differences in size, but also for measuring changes in surface conductivity, or for separations based on combinations of difference in size and dielectric properties.

  • 2.
    Aldaeus, Fredrik
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Lin, Yuan
    KTH, School of Engineering Sciences (SCI), Mechanics.
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Amberg, Gustav
    KTH, School of Engineering Sciences (SCI), Mechanics.
    Superpositioned dielectrophoresis for enhanced trapping efficiency2005In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 26, no 22, p. 4252-4259Article in journal (Refereed)
    Abstract [en]

    One of the major applications for dielectrophoresis is selective trapping and fractionation of particles. If the surrounding medium is of low conductivity, the trapping force is high, but if the conductivity increases, the attraction decreases and may even become negative. However, high-conductivity media are essential when working with biological material such as living cells. In this paper, some basic calculations have been performed, and a model has been developed which employs both positive and negative dielectrophoresis in a channel with interdigitated electrodes. The finite element method was utilized to predict the trajectories of Escherichia coli bacteria in the superpositioned electrical fields. It is shown that a drastic improvement of trapping efficiency can be obtained in this way, when a high conductivity medium is employed.

  • 3. Anderson, M. E.
    et al.
    Aslan, D.
    Clarke, A.
    Roeraade, Johan
    KTH, Superseded Departments, Chemistry.
    Hagman, G.
    Evaluation of generic chiral liquid chromatography screens for pharmaceutical analysis2003In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1005, no 02-jan, p. 83-101Article in journal (Refereed)
    Abstract [en]

    Two different automated generic liquid chromatography screens for the separation of chiral compounds of pharmaceutical interest have been evaluated. The test set comprised 53 chemically diverse chiral compounds involving 55 enantiomeric pairs from the pharmaceutical industry (i.e. starting materials, synthetic intermediates and drug substances). The first screen utilised four polysaccharide-based columns with five mobile phases and showed enantioselectivity for 87% of the test compounds. The second screen employed three macrocyclic glycopeptide columns with two mobile phases and showed enantioselectivity for 65% of the test compounds. Merging of the two screening procedures resulted in an enantioselectivity for 96% of the chiral compounds. It is anticipated that the systematic use of the automated chiral HPLC screens described in this report will substantially reduce the necessary time for method development of pharmaceutically related chiral analytical methods.

  • 4.
    Benkestock, Kurt
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Edlund, Per Olof
    Biovitrum AB, Dept Analyt Sci.
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Electrospray ionization mass spectrometry as a tool for determination of drug binding sites to human serum albumin by noncovalent interaction2005In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 19, no 12, p. 1637-1643Article in journal (Refereed)
    Abstract [en]

    Most proteins in blood plasma bind ligands. Human serum albumin (HSA) is the main transport protein with a very high capacity for binding of endogenous and exogenous compounds in plasma. Many pharmacokinetic properties of a drug depend on the level of binding to plasma proteins. This work reports studies of noncovalent interactions by means of nanoelectrospray ionization mass spectrometry (nanoESI-MS) for determination of the specific binding of selected drug candidates to HSA. Warfarin, iopanoic acid and digitoxin were chosen as site-specific probes that bind to the main sites of HSA. Two drug candidates and two known binders to HSA were analyzed using a competitive approach. The drugs were incubated with the target protein followed by addition of site-specific probes, one at a time. The drug candidates showed predominant affinity to site I (warfarin site). Naproxen and glyburide showed affinity to both sites I and II. The advantages of nanoE-SI-MS for these studies are the sensitivity, the absence of labeled molecules and the short method development time.

  • 5.
    Benkestock, Kurt
    et al.
    KTH, Superseded Departments, Chemistry.
    Edlund, Per Olof
    Biovitrum AB, Analyt Sci.
    Roeraade, Johan
    KTH, Superseded Departments, Chemistry.
    On-line microdialysis for enhanced resolution and sensitivity during electrospray mass spectrometry of non-covalent complexes and competitive binding studies2002In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 16, no 21, p. 2054-2059Article in journal (Refereed)
    Abstract [en]

    Many proteins and macromolecules easily form metal adduct ions which impairs their analysis by mass spectrometry. The present study describes how the formation of undesired adducts can be minimized by on-line microdialysis for non-covalent binding studies of macromolecules with low molecular mass ligands with electrospray ionization mass spectrometry (ESI-MS). The technique was indispensable for protein-ligand studies due to reduction of unwanted adduct ions, and thus gave excellent resolution and a sensitivity improvement of at least 5 times. The core of the analytical system was a modified microdialysis device, which was operated in countercurrent mode. A novel technique based on microdialysis for competitive binding studies is also presented. The noncovalent complex between a protein and a ligand was formed in the sample vial prior to analysis. The complex was injected into an on-line microdialysis system where a competitive ligand was administered in the dialysis buffer outside of the fiber. The second ligand competitively displaced the first ligand through transport via the wall of the dialysis fiber, and the intact complexes were detected by ESI-MS.

  • 6.
    Benkestock, Kurt
    et al.
    KTH, Superseded Departments, Chemistry.
    Sundqvist, Gustav
    KTH, Superseded Departments, Chemistry.
    Edlund, Per Olof
    Biovitrum AB, Dept Analyt Sci.
    Roeraade, Johan
    KTH, Superseded Departments, Chemistry.
    Influence of droplet size, capillary-cone distance and selected instrumental parameters for the analysis of noncovalent protein-ligand complexes by nano-electrospray ionization mass spectrometry2004In: Journal of Mass Spectrometry, ISSN 1076-5174, E-ISSN 1096-9888, Vol. 39, no 9, p. 1059-1067Article in journal (Refereed)
    Abstract [en]

    It has been suggested in the literature that nano-electrospray ionization (nano-ESI) mass spectrometry better reflects the equilibrium between complex and free protein in solution than pneumatically assisted electrospray ionization (ESI) in noncovalent interaction studies. However, no systematic studies of the effects of ionization conditions have been performed to support this statement. In the present work, different instrumental and sample-derived parameters affecting the stability of noncovalent complexes during analysis by nano-ESI were investigated. In general, increased values of parameters such as drying gas flow-rate, ion-source temperature, capillary tip voltage and buffer concentration lead to a dissociation of ribonuclease A (RNAse)-cytidine 2'-monophosphate (CMP) and cytidine 5'-triphosphate (CTP) complexes. The size of the electrosprayed droplets was shown to be an important issue. Increasing the capillary to cone distance yielded an increased complex to free protein ratio when a hydrophilic ligand was present and the reverse effect was obtained with a hydrophobic ligand. Important in this regard is the degree of sampling of ions originating from late-generation residue droplets, that is, ions present in the droplet bulk. Sampling of these ions increases with longer capillary-cone distance (flight time). Furthermore, when the sample flow-rate was increased by increasing the capillary internal tip i.d. from 4 to 30 mum, a decreased complex to free protein ratio for the RNAse-CTP system was observed. This behavior was consistent with the change in surface to volume ratio for flow-rates between 2 and 100 nl min(-1). Finally, polarity switching between positive and negative ion modes gave a higher complex to free protein ratio when the ligand and the protein had the same polarity.

  • 7.
    Benkestock, Kurt
    et al.
    KTH, Superseded Departments, Chemistry.
    Van Pelt, C. K.
    Advion BioSci Inc, Ithaca, NY USA.
    Åkerud, T.
    Lund Univ, Dept Biophys Chem.
    Sterling, A.
    Advion BioSci Ltd, Norwich, Norfolk England.
    Edlund, Per Olof
    Biovitrum AB, Analyt Sci.
    Roeraade, Johan
    KTH, Superseded Departments, Chemistry.
    Automated Nano-Electrospray Mass Spectrometry for Protein-Ligand Screening by Noncovalent Interaction Applied to Human H-FABP and A-FABP2003In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 8, no 3, p. 247-256Article in journal (Refereed)
    Abstract [en]

    A method for ligand screening by automated nano-electrospray ionization mass spectrometry (nano-ESI/MS) is described. The core of the system consisted of a chip-based platform for automated sample delivery from a 96-well plate and subsequent analysis based on noncovalent interactions. Human fatty acid binding protein, H-FABP (heart) and A-FABP (adipose), with small potential ligands was analyzed. The technique has been compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation with the found hits was obtained. In the current MS screening method, the cycle time per sample was 1.1 min, which is approximately 50 times faster than NMR for single compounds and approximately 5 times faster for compound mixtures. High reproducibility was achieved, and the protein consumption was in the range of 88 to 100 picomoles per sample. Furthermore, a novel protocol for preparation of A-FABP without the natural ligand is presented. The described screening approach is suitable for ligand screening very early in the drug discovery process before conventional high-throughput screens (HTS) are developed and/or used as a secondary screening for ligands identified by HTS.

  • 8.
    Bonn, Jonas
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Pettersson Redeby, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Electrostatic Sample Nebulization for Improved Sample Vaporization in the Split/Splitless Gas Chromatography Inlet2009In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 81, no 13, p. 5327-5332Article in journal (Refereed)
    Abstract [en]

    The split/splitless inlet system has basically the same fundamental drawbacks it had when it was introduced: poor repeatability of the injected amount of sample and discrimination of high-boiling analytes. Hot needle injection improves the repeatability of the sample transfer but suffers from in-needle discrimination. Injection with a fast autosampler, resulting in minimal heating of the needle, solves this problem but usually requires a glass wool packing in the inlet liner to assist in vaporization of the sample. As glass wool has been reported to cause degradation of labile analytes, it cannot be applied as a general remedy for improving incomplete vaporization. In this paper, a novel concept, based on electrostatic nebulization of the injected sample, is presented. The resulting fine droplets promote a more effective heat transfer and a rapid vaporization. Evaluation of the electrospray inlet in the split mode, using a straight, empty glass liner and a cold needle, showed an improvement in peak area repeatability by about 1 order of magnitude, compared with the results obtained when no electrostatic field was applied, Splitless injection of a series of hydrocarbons up to C-28 in the electrospray inlet with an empty, tapered liner, using a cold needle, showed no measurable analyte discrimination. The relative standard deviation in terms of area count for the largest hydrocarbon (C-28) was < 1.5%, compared to similar to 30% for injections where no high voltage was applied.

  • 9.
    Bonn, Jonas
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Redeby, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    A novel injection technique for the split/splitless gas chromatography inletManuscript (Other academic)
  • 10.
    Bonn, Jonas
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Redeby, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Mixed sorbent phases for thick film open tubular traps2009In: Journal of Chromatographic Science, ISSN 0021-9665, E-ISSN 1945-239X, Vol. 47, no 4, p. 297-303Article in journal (Refereed)
    Abstract [en]

    In this work, we present a technique for the preparation of tailor-made sorbent phases for thick film open tubular traps. Solid or liquid polymers are dispersed in a polydimethylsiloxane (PDMS) pre-polymer, which is cross-linked in situ after coating. The technique is evaluated by preparing thick film open tubular traps with PDMS containing solid or liquid poly(ethylene glycol) (PEG). A significant increase in retention for polar analytes is observed, even when only 7.5% PEG is present. The increase in retention for 3-chloro-1,2-propanediol is more than tenfold. The preparation method is simple and no solvents are required. Also, the concept provides great flexibility in terms of phase composition.

  • 11. Chabert, Max
    et al.
    Dorfman, Kevin D.
    de Cremoux, Patricia
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Viovy, Jean-Louis
    Automated microdroplet platform for sample manipulation and polymerase chain reaction2006In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 78, no 22, p. 7722-7728Article in journal (Refereed)
    Abstract [en]

    We present a fully automated system performing continuous sampling, reagent mixing, and polymerase chain reaction (PCR) in microdroplets transported in immiscible oil. Sample preparation and analysis are totally automated, using an original injection method from a modified 96-well plate layered with three superimposed liquid layers and in-capillary laser-induced fluorescence endpoint detection. The process is continuous, allowing sample droplets to be carried uninterruptedly into the reaction zone while new drops are aspirated from the sample plate. Reproducible amplification, negligible cross-contamination, and detection of low sample concentrations were demonstrated on numerous consecutive sample drops. The system, which opens the route to strong reagents and labor savings in high-throughput applications, was validated on the clinically relevant quantification of progesterone receptor gene expression in human breast cancer cell lines.

  • 12. Chilo, J.
    et al.
    Horvath, G.
    Lindblad, Thomas
    KTH, School of Engineering Sciences (SCI), Physics.
    Olsson, R.
    Redeby, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry (closed 20110630).
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry (closed 20110630).
    A flexible electronic nose for odor discrimination using different methods of classification2009In: 2009 16th IEEE-NPSS Real Time Conference - Conference Record, 2009, p. 317-320Conference paper (Refereed)
    Abstract [en]

    Ovarian cancer is one of the leading causes of death from cancer in women. The lifetime risk is around 1.5%, which makes it the second most common gynecologic malignancy (the first one being breast cancer). To have a definitive diagnose, a surgical procedure is generally required and suspicious areas (samples) will be removed and sent for microscopic and other analysis. This paper describes the result of a pilot study in which an electronic nose is used to "smell" the aforementioned samples, analyze the multi-sensor signals and have a close to real-time answer on the detection of cancer. Besides being fast, the detection method is inexpensive and simple. Experimental analysis using real ovarian carcinoma samples shows that the use of proper algorithms for analysis of the multi-sensor data from the electronic nose yielded surprisingly good results with more than 77% classification rate. The electronic nose used in this pilot study was originally developed to be used as a "bomb dog" and can distinguish between e.g. TNT, Dynamex, Prillit. However, it was constructed to be a flexible multi-sensor device and the individual (16) sensors can easily be replaced/exchanged. This is suggestive for further investigations to obtain even better results with new, specific sensors. In another pilot experiment, headspace of an ovarian carcinoma sample and a control sample were analyzed using gas chromatography-mass spectrometry. Significant differences in chemical composition and compound levels were recorded, which would explain the different response obtained with the electronic nose.

  • 13. Curcio, M.
    et al.
    Roeraade, Johan
    KTH, Superseded Departments, Chemistry.
    Continuous segmented-flow polymerase chain reaction for high-throughput miniaturized DNA amplification2003In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 75, no 1, p. 1-7Article in journal (Refereed)
    Abstract [en]

    A continuous segmented-flow method for sequential DNA amplification is described in order to provide a basis for high-throughput genetic analysis. The approach allows an immediate distinction between amplified and nonamplified products. A mixture of sample and reagents are loaded in the form of small segments one after another in a 15-m-long narrow-bore Teflon tube, coiled such as to be repeatedly exposed to three different temperature zones. After having passed the heated zones, the samples are mixed with an intercalating dye by flow injection and sequentially detected on-line by laser-induced fluorescence. The aqueous samples travel as separate segments in a continuous flow of an immiscible, organic. liquid. Perfluorodecalin was shown to be particularly suitable due to its hydrophobicity and inert properties. To reduce carryover between samples, an intermediate water plug between two consecutive samples was required. Selected regions from human genomic DNA were successfully amplified in 300-nL volumes after 30 passes through the heated zones. The total reaction time was similar to45 min, and the detection interval between individual samples was 1 min. Automation and the possibility to further reduce sample volumes, as well as to employ many reaction columns simultaneously, should provide a platform for an extremely high throughput.

  • 14. Curcio, M.
    et al.
    Stalhandske, P.
    Lindberg, P.
    Roeraade, Johan
    KTH, Superseded Departments, Chemistry.
    Multiplex high-throughput solid-phase minisequencing by capillary electrophoresis and liquid core waveguide fluorescence detection2002In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 23, no 10, p. 1467-1472Article in journal (Refereed)
    Abstract [en]

    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.

  • 15.
    Ek, Patrik
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    New Method for Fabrication of Fused Silica Emitters with Submicrometer Orifices for Nanoelectrospray Mass Spectrometry2011In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 83, no 20, p. 7771-7777Article in journal (Refereed)
    Abstract [en]

    In this paper, we describe a new method for fabrication of nanoelectrospray emitters. The needles were pulled from fused silica capillary tubing, which was melted by means of a plasma, formed by electrical discharges between two pointed platinum electrodes. A key feature of the pulling device is a rotating configuration of the electrodes, which results in an even radial heating of the capillary. The construction of the setup is straightforward, and needles with a variety of shapes can be fabricated, including orifices of submicrometer dimensions. Pulled needles with long tapered tips and an orifice of 0.5 mu m were utilized for electrospray ionization mass spectrometry (ESI-MS) of discrete sample volumes down to 275 pL. The picoliter-sized samples were transferred into the tip of the needle from a silicon microchip by aspiration. To avoid a rapid evaporation of the sample, all manipulations were performed under a cover of a fluorocarbon liquid. The limit of detection was measured to be ca. 20 attomole for insulin (chain B, oxidized).

  • 16.
    Ek, Patrik
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Schönberg, Tommy
    Sjödahl, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Jacksén, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Vieider, Christian
    Emmer, Åsa
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Electrospray Ionization from an Adjustable Gap between two Silicon Chips2009In: Journal of Mass Spectrometry, ISSN 1076-5174, E-ISSN 1096-9888, Vol. 44, no 2, p. 171-181Article in journal (Refereed)
    Abstract [en]

    In this paper, a silicon chip - based electrospray emitter with a variable orifice size is presented. The device consists of two chips, with a thin beam elevating from the center of each of the chips. The chips are individually mounted to form an open gap of a narrow, uniform width between the top areas of the beams. The electrospray is generated at the endpoint of the gap, where the spray point is formed by the very sharp intersection between the crystal planes of the < 100 > silicon chips. Sample solution is applied to the rear end of the gap from a capillary via a liquid bridge, and capillary forces ensure a spontaneous imbibition of the gap. The sample solution is confined to the gap by means of a hydrophobic treatment of the surfaces surrounding the gap, as well as the geometrical boundaries formed by the edges of the gap walls. The gap width could be adjusted between 1 and 25 μm during electrospray experiments without suffering from any interruption of the electrospray process. Using a peptide sample solution, a shift toward higher charge states and increased signal-to-noise ratios was observed when the gap width was decreased. The limit of detection for the peptide insulin (chain B, oxidized) was approximately 4 nM. We also show a successful interfacing of the electrospray setup with capillary electrophoresis.

  • 17.
    Ek, Patrik
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Sjödahl, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Electrospray Ionization from a Gap with Adjustable Width2006In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 20, no 21, p. 3176-3182Article in journal (Refereed)
    Abstract [en]

    In this paper, we present a new concept for electrospray ionization mass spectrometry, where the sample is applied in a gap which is formed between the edges of two triangular-shaped tips. The size of the spray orifice can be changed by varying the gap width. The tips were fabricated from polyethylene terephthalate film with a thickness of 36 μm. To improve the wetting of the gap and sample confinement, the edges of the tips forming the gap were hydrophilized by means of silicon dioxide deposition. Electrospray was performed with gap widths between 1 and 36 μm and flow rates down to 75 nL/min. The gap width could be adjusted in situ during the mass spectrometry experiments and nozzle clogging could be managed by simply widening the gap. Using angiotensin I as analyte, the signal-to-noise ratio increased as the gap width was decreased, and a shift towards higher charge states was observed. The detection limit for angiotensin I was in the low nM range.

  • 18.
    Ek, Patrik
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry (closed 20110630).
    Stjernström, Mårten
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry (closed 20110630).
    Emmer, Åsa
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry (closed 20110630).
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry (closed 20110630).
    Electrospray ionization mass spectrometry from discrete nanoliter-sized sample volumes2010In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 24, no 17, p. 2561-2568Article in journal (Refereed)
    Abstract [en]

    We describe a method for nanoelectrospray ionization mass spectrometry (nESI-MS) of very small sample volumes. Nanoliter-sized sample droplets were taken up by suction into a nanoelectrospray needle from a silicon microchip prior to ESI. To avoid a rapid evaporation of the small sample volumes, all manipulation steps were performed under a cover of fluorocarbon liquid. Sample volumes down to 1.5 nL were successfully analyzed, and an absolute limit of detection of 105 attomole of insulin (chain B, oxidized) was obtained. The open access to the sample droplets on the silicon chip provides the possibility to add reagents to the sample droplets and perform chemical reactions under an extended period of time. This was demonstrated in an example where we performed a tryptic digestion of cytochrome C in a nanoliter-sized sample volume for 2.5h, followed by monitoring the outcome of the reaction with nESI-MS. The technology was also utilized for tandem mass spectrometry (MS/MS) sequencing analysis of a 2 nL solution of angiotensin I.

  • 19.
    Emmer, Åsa
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Enzymatic protein digest in chip-based nanovials with immobilized proteolytic enzymes2005In: Analytica Chimica Acta, ISSN 0003-2670, E-ISSN 1873-4324, Vol. 542, no 2, p. 137-143Article in journal (Refereed)
    Abstract [en]

    In the present work, protein digest reactions in silicon-based microchips, coated with immobilized proteolytic enzymes, have been carried out. The performance of such vials, modified with trypsin or chymotrypsin, was tested with myoglobin as a substrate. Capillary electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry were utilized for analysis of the digests, and the influence of different instrumentation setups. immobilization procedures and reaction conditions are discussed.

  • 20.
    Emmer, Åsa
    et al.
    KTH, Superseded Departments, Chemistry.
    Roeraade, Johan
    KTH, Superseded Departments, Chemistry.
    Wall deactivation with fluorosurfactants for capillary electrophoretic analysis of biomolecules2001In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 22, no 4, p. 660-665Article in journal (Refereed)
    Abstract [en]

    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.

  • 21.
    Gantelius, Jesper
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Hartmann, Michael
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics.
    Roeraade, Johan
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Joos, Thomas O
    Magnetic bead-based detection of autoimmune responses using protein microarrays.2009In: New biotechnology, ISSN 1871-6784, Vol. 26, p. 269-276Article in journal (Refereed)
    Abstract [en]

    In the present study, a magnetic bead-based detection approach for protein microarrays is described as an alternative approach to the commonly used fluorescence-based detection system. Using the bead-based detection approach with applied magnetic force, it was possible to perform the detection step more rapidly as a result of the accelerated binding between the captured analyte in the microspot and the detection antibody, which was coupled to the magnetic beads. The resulting strong opacity shift on the microspots could be recorded with an ordinary flatbed scanner. In the context of autoimmunity, a set of 24 serum samples was analyzed for the presence of antibodies against 12 autoantigens using standard fluorescence and magnetic bead-based detection methods. Dynamic range, sensitivity, and specificity were determined for both detection methods. We propose from our findings that the magnetic bead-based detection option provides a simplified and cost effective readout method for protein microarrays.

  • 22. Griss, P.
    et al.
    Melin, J.
    Sjodahl, J.
    Roeraade, Johan
    KTH, Superseded Departments, Chemistry.
    Stemme, Göran
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Development of micromachined hollow tips for protein analysis based on nanoelectrospray ionization mass spectrometry2002In: Journal of Micromechanics and Microengineering, ISSN 0960-1317, E-ISSN 1361-6439, Vol. 12, no 5, p. 682-687Article in journal (Refereed)
    Abstract [en]

    Two novel types of micromachined nanoelectrospray emitter tips have been designed, fabricated and tested. The fabrication method of the hollow tips is based on a self-aligning deep reactive ion etch process. The tips consist of either silicon dioxide or silicon and feature orifice diameters of 10 and 18 mum, respectively. The geometrical characteristics of both emitter types are favorable for the generation of stable electrospray ionization, i.e. wetting of the tip shaft is avoided and the base of the Taylor cone is limited to the diameter of the orifice. A silicon dioxide tip was operated in a bench top setup to visually evaluate the electrospray. Both types of tips were also successfully used for the analysis of an insulin sample in an ion trap mass spectrometer.

  • 23.
    Hamberg, Anders
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Kempka, Martin
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Sjödahl, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    C-terminal ladder sequencing of peptides using an alternative nucleophile in carboxypeptidase Y digests2006In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 357, no 2, p. 167-172Article in journal (Refereed)
    Abstract [en]

     A method for improved sequence coverage in C-terminal sequencing of peptides, based on carboxypeptidase digestion, is described. In conventional carboxypeptidase digestions, the peptide substrate is usually extensively degraded and a full amino acid sequence cannot be obtained due to the lack of a complete peptide ladder. In the presented method, a protecting group is introduced at the C terminus of a fraction of the peptide fragments formed in the digest, and thereby further degradation of the C-terminally modified peptides are slowed down. The protecting group was attached to the C-terminal amino acid through a carboxypeptidase-catalyzed reaction with an alternative nucleophile, 2-pyridylmethylamine, added to the aqueous digestion buffer. Six peptides were digested by carboxypeptidase Y with and without 2-pyridylmethylamine present in the digest buffer, and the resulting fragments subsequently were analyzed with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Comparison of the two digestion methods showed that the probability of successful ladder sequencing increased, by more than 50% using 2-pyridylmethylamine as a competing nucleophile in carboxypeptidase Y digests.

  • 24. Hanning, A.
    et al.
    Lindberg, P.
    Westberg, J.
    Roeraade, Johan
    KTH, Superseded Departments, Chemistry.
    Laser induced fluorescence detection by liquid core waveguiding applied to DNA sequencing by capillary electrophoresis2000In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 72, no 15, p. 3423-3430Article in journal (Refereed)
    Abstract [en]

    A new laser-induced fluorescence detector for capillary electrophoresis (CE) is described. The detector is based on transverse illumination and collection of the emitted fluorescent light via total internal reflection along the separation capillary. The capillary is coated with a low refractive index fluoropolymer and serves as a liquid core waveguide (LCW). The emitted light is detected end-on with a CCD camera at the capillary exit. The observed detection limit for fluorescein is 2.7 pM (550 ymol) in the continuous-flow mode and 62 fM in the CE mode. The detector is applied to DNA sequencing. One-color G sequencing is performed with single-base resolution and signal-to-noise ratio similar to 250 for peaks around 500 bases. The signal-to-noise ratio is similar to 50 for peaks around 950 bases. Full four-color DNA sequencing is also demonstrated. The high sensitivity of the detector is suggested to partly be due to the efficient rejection of scattered laser light in the LCW. The concept should be highly suitable for capillary array detection.

  • 25. Hanning, A.
    et al.
    Westberg, J.
    Roeraade, Johan
    KTH, Superseded Departments, Chemistry.
    A liquid core waveguide fluorescence detector for multicapillary electrophoresis applied to DNA sequencing in a 91-capillary array2000In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 21, no 15, p. 3290-3304Article in journal (Refereed)
    Abstract [en]

    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.

  • 26.
    Hartmann, Michael
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Stoll, Dieter
    Templin, Markus F.
    Joos, Thomas O.
    Protein microarrays for diagnostic assays2009In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 393, no 5, p. 1407-1416Article in journal (Refereed)
    Abstract [en]

    Protein microarray technology has enormous potential for in vitro diagnostics (IVD). Miniaturized parallelized immunoassays are perfectly suited to generating a maximum of diagnostically relevant information from minute amounts of sample whilst only requiring small amounts of reagent. Protein microarrays have become well-established research tools in basic and applied research and the first products are already on the market. This article reviews the current state of protein microarrays and discusses developments and future demands relating to protein arrays in their role as multiplexed immunoassays in the field of diagnostics.

  • 27.
    Hartmann, Michael
    et al.
    NMI–Natural and Medical Sciences Institute at the University of Tübingen.
    Schrenk, Monika
    Dottinger, Anette
    Nagel, Sarah
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Joos, Thomas O.
    Templin, Markus F.
    Expanding assay dynamics: A combined competitive and direct assay system for the quantification of proteins in multiplexed Immunoassays2008In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 54, no 6, p. 956-963Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: The concurrent detection and quantification of analytes that vary widely in concentration present a principal problem in multiplexed assay systems. Combining competitive and sandwich immunoassays permits coverage of a wide concentration range, and both highly abundant molecules and analytes present in low concentration can be quantified within the same assay. METHODS: The use of different fluorescence readout channels allows the parallel use of a competitive system and a sandwich configuration. The 2 generated assay signals are combined and used to calculate the amount of analyte. The measurement range can be adjusted by varying the competitor concentration, and an extension of the assay system's dynamic range is possible. RESULTS: We implemented the method in a planar protein microarray-based autoimmune assay to detect autoantibodies against 13 autoantigens and to measure the concentration of a highly abundant protein, total human IgG, in one assay. Our results for autoantibody detection and IgG quantification agreed with results obtained with commercially available assays. The use of 2 readout channels in the protein microarray-based system reduced spot-to-spot variation and intraassay variation. CONCLUSIONS: By combining a direct immunoassay with a competitive system, analytes present in widely varying concentrations can be quantified within a single multiplex assay. Introducing a second readout channel for analyte quantification is an effective tool for spot-to-spot normalization and helps to lower intraassay variation.

  • 28.
    Hartmann, Michael
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Sjödahl, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Stjernström, Mårten
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Pettersson Redeby, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Joos, Thomas
    NMI–Natural and Medical Sciences Institute at the University of Tübingen.
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Non-contact protein microarray fabrication using a procedure based on liquid bridge formation2009In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 393, no 2, p. 591-598Article in journal (Refereed)
    Abstract [en]

    Contemporary microarrayers of contact or noncontact format used in protein microarray fabrication still suffer from a number of problems, e. g. generation of satellite spots, inhomogeneous spots, misplaced or even absent spots, and sample carryover. In this paper, a new concept of non-contact sample deposition that reduces such problems is introduced. To show the potential and robustness of this pressure-assisted deposition technique, different sample solutions known to cause severe problems or to be even impossible to print with conventional microarrayers were accurately printed. The samples included 200 mg mL(-1) human serum albumin, highly concentrated sticky cell adhesion proteins, pure high-salt cell-lysis buffer, pure DMSO, and a suspension of 5-mu m polystyrene beads. Additionally, a water-immiscible liquid fluorocarbon, which was shown not to affect the functionality of the capture molecules, was employed as a lid to reduce evaporation during microarray printing. The fluorocarbon liquid lid was shown to circumvent hydrolysis of water-sensitive activated surfaces during long-term deposition procedures.

  • 29.
    Hult, E L
    et al.
    KTH, Superseded Departments, Chemistry.
    Emmer, Åsa
    KTH, Superseded Departments, Chemistry.
    Roeraade, Johan
    KTH, Superseded Departments, Chemistry.
    Capillary electrophoretic separation of acidic and basic proteins in the presence of cationic and anionic fluorosurfactants1997In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 757, no 1-2, p. 255-262Article in journal (Refereed)
  • 30. Johansson, L.
    et al.
    Aldaeus, Fredrik
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Jonsson, C.
    Hamp, S.
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE).
    Determination of conductivity of bacteria by using cross-flow filtration2006In: Biotechnology letters, ISSN 0141-5492, E-ISSN 1573-6776, Vol. 28, no 8, p. 601-603Article in journal (Refereed)
    Abstract [en]

    An important property of the bacterial surface is its conductivity. To obtain reliable conductivity values, it is essential to handle the cells as gently as possible during the measurement procedure. We have developed a method where a standard conductivity meter is used in combination with cross-flow filtration, thus avoiding repeated centrifugation and resuspension. With this method, the conductivity of Bacillus subtilis was determined to be 7000 mu S/cm, which is a deviation from previously published data by almost an order of a magnitude.

  • 31.
    Kempka, Martin
    et al.
    KTH, Superseded Departments, Chemistry.
    Sjödahl, Johan
    KTH, Superseded Departments, Chemistry.
    Björk, Anders
    KTH, Superseded Departments, Chemistry.
    Roeraade, Johan
    KTH, Superseded Departments, Chemistry.
    Improved method for peak picking in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry2004In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 18, no 11, p. 1208-1212Article in journal (Refereed)
    Abstract [en]

    A method for peak picking for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is described. The method is based on the assumption that two sets of ions are formed during the ionization stage, which have Gaussian distributions but different velocity profiles. This gives rise to a certain degree of peak skewness. Our algorithm deconvolutes the peak and utilizes the fast velocity, bulk ion distribution for peak picking. Evaluation of the performance of the new method was conducted using peptide peaks from a bovine serum albumin (BSA) digest, and compared with the commercial peak-picking algorithms Centroid and SNAP. When using the new two-Gaussian algorithm, for strong signals the mass accuracy was equal to or marginally better than the results obtained from the commercial algorithms. However, for weak, distorted peaks, considerable improvement in both mass accuracy and precision was obtained. This improvement should be particularly useful in proteomics, where a lack of signal strength is often encountered when dealing with weakly expressed proteins. Finally, since the new peak-picking method uses information from the entire signal, no adjustments of parameters related to peak height have to be made, which simplifies its practical use.

  • 32.
    Kloskowski, Adam
    et al.
    KTH, Superseded Departments, Chemistry.
    Pettersson, Johan
    KTH, Superseded Departments, Chemistry.
    Roeraade, Johan
    KTH, Superseded Departments, Chemistry.
    Thick film traps with an irregular film: Preparation and evaluation2004In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1035, no 2, p. 159-165Article in journal (Refereed)
    Abstract [en]

    A new method for preparation of sorbent-based ultra-thick film traps for concentration of trace volatile components from gaseous matrices is described. The procedure is based on blowing a prepolymer (polydimethylsiloxane) through a capillary tube, forming an irregular film of stationary phase. Subsequently, the prepolymer is immobilized in a few seconds by heating to 200 °C. Evaluation of the performance of the new traps showed that the loss of efficiency, compared to regular smooth film traps is only on the order of 20–30%. In terms of breakthrough volume, this loss in performance is rather insignificant. The technology is extremely simple and allows a rapid and cheap production of a large number of ultra-thick film traps, even in non-specialized laboratories. The method can be applied to any type of cross-linkable stationary phase, thereby expanding the scope of sorbent-based trapping and preconcentration concept. Many applications are anticipated in trace and ultra-trace analysis in a wide range of fields, such as environmental chemistry, polymers, food and process analysis.

  • 33.
    Lin, Yuan
    et al.
    KTH, School of Engineering Sciences (SCI), Mechanics.
    Amberg, Gustav
    KTH, School of Engineering Sciences (SCI), Mechanics.
    Aldaeus, Fredrik
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry (closed 20110630).
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry (closed 20110630).
    Simulation of dielectrophoretic motion of microparticles using a molecular dynamics approach2006In: 4th International Conference on Nanochannels, Microchannels and Minichannels, ICNMM2006, 2006, p. 1-10Conference paper (Refereed)
    Abstract [en]

    We model and simulate dielectrophoresis of microscale particles using the finite element method. A soft sphere system molecular dynamics model is presented, which solves a set of equations for the motion of every particle. The model couples most of the significant forces, i.e. the dielectrophoresis (DEP) forces, the particle-particle electrostatic forces, particle-particle interfacial repulsive forces, particle-wall repulsive forces and the hydrodynamic forces in Stokes flow. Since the system of equations is stiff, an implicit scheme is used. To obtain the particle trajectories, a constant time-step is applied. We present some numerical tests computing hydrodynamic force, electrostatic force and DEP force using our model, including simulated trapping of particles in a micro channel by dielectrophoresis. The results are in agreement with the theories and the experimental observations.

  • 34. Litborn, E.
    et al.
    Emmer, Åsa
    KTH, Superseded Departments, Chemistry.
    Roeraade, Johan
    KTH, Superseded Departments, Chemistry.
    Parallel reactions in open chip-based nanovials with continuous compensation for solvent evaporation2000In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 21, no 1, p. 91-99Article in journal (Refereed)
    Abstract [en]

    In an earlier report (Litborn, E., Emmer,,Angstrom., Roeraade, J., Anal. Chim. Acta 1999, 401, 11-19, we described a technique for performing chemistry in chip-based vials. A major problem, solvent evaporation, was partially remedied by using a closed humidity chamber. in this paper we report an improved technique for performing parallel reactions in open, 15 nL volume, chip-based vials. The evaporation of solvent from the reaction fluid was continuously compensated by addition of solvent via an array of microcapillaries. The suitability of the method was demonstrated by performing eight separate peptide maps of myoglobin in parallel, using the three enzymes trypsin, alpha-chymotrypsin and endoproteinase Glu-C. The total amount of myoglobin utilized to perform the eight digests was less than 100 pmol. The corresponding amount of enzymes was ca. 0.1 pmol per reaction. In order to evaluate the operating limits of the technique, a study of the evaporation of solvents from a series of vials with proportionally smaller volumes operated at different temperatures was performed. The results showed that the concept for continuous compensation of solvent evaporation should be applicable to reaction volumes down to 30 pL.

  • 35. Litborn, E.
    et al.
    Roeraade, Johan
    KTH, Superseded Departments, Chemistry.
    Liquid lid for biochemical reactions in chip-based nanovials2000In: Journal of Chromatography B, ISSN 0378-4347, Vol. 745, no 1, p. 137-147Article in journal (Refereed)
    Abstract [en]

    The present paper describes a new technique to suppress evaporation of solvent from very small volumes. Vials (15 nl) on a silicon microchip were filled with water, and covered with a thin, flowing film of a volatile liquid (e.g., octane). Water evaporation was greatly reduced. At 37 degrees C, no appreciable loss of water could be observed over a period of 90 min. At 95 degrees C, most of the water sample was left in the vial for more than 3 min. The applicability of the method is demonstrated by a tryptic digest, where the resulting peptide fragments were analyzed by capillary electrophoresis. In addition, a discussion of the possibilities and limitations of some alternative methods is included in the paper, as well as an outlook on future developments of the liquid lid concept.

  • 36.
    Mikkonen, Saara
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry.
    Jacksén, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry.
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry.
    Thormann, Wolfgang
    University of Bern, Clinical Pharmacology Laboratory, Institute for Infectious Diseases.
    Emmer, Åsa
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry.
    Microfluidic Isoelectric Focusing of Amyloid Beta Peptides Followed by Micropillar-Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry2016In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882Article in journal (Refereed)
    Abstract [en]

    A novel method for preconcentration and purification of the Alzheimer’s disease related amyloid beta (Aβ) peptides by isoelectric focusing (IEF) in 75 nL microchannels combined with their analysis by micropillar-matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) is presented. A semiopen chip-based setup, consisting of open microchannels covered by a lid of a liquid fluorocarbon, was used. IEF was performed in a mixture of four small and chemically well-defined amphoteric carriers, glutamic acid, aspartyl-histidine (Asp-His), cycloserine (cSer), and arginine, which provided a stepwise pH gradient tailored for focusing of the C-terminal Aβ peptides with a pI of 5.3 in the boundary between cSer and Asp-His. Information about the focusing dynamics and location of the foci of Aβ peptides and other compounds was obtained using computer simulation and by performing MALDI-MS analysis directly from the open microchannel. With the established configuration, detection was performed by direct sampling of a nanoliter volume containing the focused Aβ peptides from the microchannel, followed by deposition of this volume onto a chip with micropillar MALDI targets. In addition to purification, IEF preconcentration provides at least a 10-fold increase of the MALDI-MS-signal. After immunoprecipitation and concentration of the eluate in the microchannel, IEF-micropillar-MALDI-MS is demonstrated to be a suitable platform for detection of Aβ peptides in human cerebrospinal fluid as well as in blood plasma.

  • 37.
    Pettersson, Johan
    et al.
    KTH, Superseded Departments, Chemistry.
    Aldaeus, Fredrik
    KTH, Superseded Departments, Chemistry.
    Kloskowski, Adam
    KTH, Superseded Departments, Chemistry.
    Roeraade, Johan
    KTH, Superseded Departments, Chemistry.
    Ultra thick film open tubular traps with an increased inner diameter2004In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1047, no 1, p. 93-99Article in journal (Refereed)
    Abstract [en]

    In this paper, the concept of open tubular traps, coated with a very thick film of polydimethyldisiloxane for enrichment of trace volatile components has been further explored. From theoretical calculations as well as practical experiments it is demonstrated that it can be advantageous to increase the inner diameter of such traps. For a given sampling flow rate and phase ratio, the plate number of the traps is not dependent on the inner diameter, provided that the linear flow velocity remains sufficiently high to offset the effect of axial diffusion. It is shown that this is due to the basic fact that for a given sampling flow rate, the average linear flow velocity in the trap is inversely proportional to the square of the inner diameter of the trap. However, in contrast to chromatographic separations, the linear flow velocity is not important. Under conditions of a constant phase ratio, an increased inner diameter also increases the amount of sorbent in the trap, which is a key parameter for obtaining high breakthrough volumes. Open tubular traps with an expanded inner diameter have very low pressure drop characteristics, which provides the possibility to construct new, simplified sampling systems.

  • 38.
    Pettersson, Johan
    et al.
    KTH, Superseded Departments, Chemistry.
    Kloskowski, Adam
    KTH, Superseded Departments, Chemistry.
    Zaniol, Carlo
    KTH, Superseded Departments, Chemistry.
    Roeraade, Johan
    KTH, Superseded Departments, Chemistry.
    Automated high-capacity sorption probe for extraction of organic compounds in aqueous samples followed by gas chromatographic analysis2004In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1033, no 2, p. 339-347Article in journal (Refereed)
    Abstract [en]

    An automated high-capacity sorption device for GC analysis of ultra trace components has been developed. The scope of the presented technique was to combine the simplicity of solid-phase microextraction (SPME) with the high extraction efficiency of the stir bar sorptive extraction technology. Sorptive extractions of water samples were performed using polydimethylsiloxane (PDMS) rubber tubing (120 μl) mounted onto a glass rod. The sampling procedure was carried out by a robotic autoinjector. Since the setup is fully automated, unattended and precise time-controlled extraction of samples is possible and makes quantitation with non-equilibrium extractions feasible. The sorption probes are easy to exchange, which facilitates off-line/in-field sampling. The system was evaluated with a test mixture of 44 environmentally hazardous compounds. Detection limits were found to be in the sub-ppt region. The performance of the system was demonstrated with the analysis of polycyclic aromatic hydrocarbons in urban snow.

  • 39.
    Pettersson, Johan
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Method for analysis of polar volatile trace components in aqueous samples by gas chromatography2005In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 77, no 10, p. 3365-3371Article in journal (Refereed)
    Abstract [en]

    A new method has been developed for direct analysis of volatile polar trace compounds in aqueous samples by gas chromatography. Water samples are injected onto a short packed precolumn containing anhydrous lithium chloride. A capillary column is coupled in series with the prefractionation column for final separation of the analytes. The enrichment principle of the salt precolumn is reverse to the principles employed in conventional methods such as SPE or SPME in which a sorbent or adsorbent is utilized to trap or concentrate the analytes. Such methods are not efficient for highly polar compounds. In the LiCl pre-column concept, the water matrix is strongly retained on the hygroscopic salt, whereas polar as well as nonpolar volatile organic compounds show very low retention and are eluted ahead of the water. After transfer of the analytes to the capillary column, the retained bulk water is removed by backflushing the precolumn at elevated temperature. For direct injections of 120 μL of aqueous samples, the combined time for injection and preseparation is only 3.5 min. With this procedure, direct repetitive automated analyses of highly volatile polar compounds such as methanol or tetrahydrofuran can be performed, and a limit of quantification in the low parts-per-billion region utilizing a flame ionization detector is demonstrated.

  • 40.
    Pettersson, Johan
    et al.
    KTH, Superseded Departments, Chemistry.
    Roeraade, Johan
    KTH, Superseded Departments, Chemistry.
    Quantitative accuracy in the gas chromatographic analysis of solvent mixtures2003In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 985, no 1-2, p. 21-27Article in journal (Refereed)
    Abstract [en]

    Quantitative accuracy is of great importance in the analysis of bulk mixtures of solvents, particularly when the analysis is related to quality control of very large product volumes like in solvent recovery plants. Serious errors can be made if the effects of density differences between the pure solvents and volume contractions are not properly addressed. In earlier work, the use of an iterative process for correcting such errors has been suggested. However, in the case of volume contractions and mixtures of several solvents, this procedure is difficult to apply. In the present paper, we describe a simple procedure where calibration curves based on mass concentration are utilized. The densities of calibration mixtures of known compositions are determined with a density meter, in order to provide for correction factors caused by volume contractions. Model experiments with mixtures of water, ethanol, acetone and methanol showed a significant improvement in quantitative accuracy, when the suggested calibration strategy was applied.

  • 41.
    Redeby, Theres
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry (closed 20110630).
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry (closed 20110630).
    Emmer, Åsa
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry (closed 20110630).
    Simple fabrication of a structured matrix-assisted laser desorption/ionization target coating for increased sensitivity in mass spectrometric analysis of membrane proteins2004In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 18, no 10, p. 1161-1166Article in journal (Refereed)
    Abstract [en]

    A new prestructured target plate for matrix-assisted laser desorption/ionization (MALDI) was developed specifically for hydrophobic integral membrane proteins. This sample support contains predefined concentrating sample spots with a focusing effect on droplets with a high content of hexafluoroisopropanol (HFIP). This fluorinated organic solvent is advantageous for solubilizing hydrophobic proteins that are not soluble in water or the organic solvents normally used in sample preparation protocols for MALDI-MS. The prestructured plate was constructed by coating a regular steel plate with a thin layer of a silicone polymer, leaving sample spots of bare steel. Fabrication of the concentrating silicone structure was fast and very straightforward, without expensive or complicated equipment. Removing the layer, and thus regenerating the steel plate, was done by a simple washing procedure. The application and cleaning procedure are not constrained by a particular design of sample support or to any specific brand of mass spectrometer. When using the prestructured MALDI plate with HFIP as the sample solvent for 17 pmol of a cyanogen bromide digest of the highly hydrophobic membrane protein bacteriorhodopsin, an improved focusing effect and an increase of more than five-fold in average sensitivity were observed, compared with a regular steel target. Experimental results show a two-fold increase in average sensitivity when the new prestructured target plate was used, compared with a commercially available concentrating support

  • 42. Rice, M.
    et al.
    Roeraade, Johan
    KTH, Superseded Departments, Chemistry.
    Continuous filtration and titration apparatus for real time monitoring of polyelectrolyte concentration and cationic demand of a paper furnish2003In: Nordic Pulp & Paper Research Journal, ISSN 0283-2631, E-ISSN 2000-0669, Vol. 18, no 1, p. 95-107Article in journal (Refereed)
    Abstract [en]

    This paper presents the application of a continuous real-time colorimetric titrator coupled to a continuous filtration apparatus, allowing polyelectrolyte concentration and cationic demand measurements to be undertaken on a paper furnish. A continuous filtration device is utilized that selectively separates dissolved and colloidal substances (DCS) from a paper furnish. By utilizing a back titration procedure, it was possible to utilize the continuous titration apparatus directly on the filtrate in order to monitor furnish charge on a pulp sample in real-time. The apparatus was utilized to study the dynamics of polyelectrolyte adsorption onto fiber surfaces, as well the change of fiber surface charge at varying pH values and for different pulp samples taken from the bleaching process of a kraft pulp.

  • 43.
    Roeraade, Johan
    KTH, Superseded Departments, Chemistry.
    New strategies for analysis of polar organic ultra trace components in water2003In: Chimia (Basel), ISSN 0009-4293, Vol. 57, no 02-jan, p. 10-11Article in journal (Refereed)
  • 44.
    Roeraade, Johan
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry.
    Stjernström, Mårten
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry.
    Emmer, Åsa
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry.
    Litborn, Erik
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry.
    Lindberg, Ulf
    Uppsala Universitet.
    Nanochemistry and nanoseparations of biomolecules1996In: microTAS, Special issue - Analytical methods & instrumentation, p. 26-30Article in journal (Refereed)
  • 45. Sjodahl, J
    et al.
    Emmer, A
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry.
    Karlstam, B
    Vincent, J
    Roeraade, J
    Separation of proteolytic enzymes originating from Antarctic krill (Euphausia superba) by capillary electrophoresis1998In: JOURNAL OF CHROMATOGRAPHY B, ISSN 0378-4347, Vol. 705, no 2, p. 231-241Article in journal (Refereed)
  • 46. Sjodahl, J.
    et al.
    Emmer, Åsa
    KTH, Superseded Departments, Chemistry.
    Vincent, J.
    Roeraade, Johan
    KTH, Superseded Departments, Chemistry.
    Characterization of proteinases from Antarctic krill (Euphausia superba)2002In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 26, no 1, p. 153-161Article in journal (Refereed)
    Abstract [en]

    Fractions of three trypsin-like proteinases, TL I, TL II, and TL III, a chymotrypsin-like proteinase, CL, two carboxypeptidase A enzymes, CPA I and CPA II and two carboxypeptidase B enzymes. CPB I and CPB II, from Antarctic krill (Euphausia superba) have been characterized with respect to purity by the means of capillary electrophoresis, CE, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The masses of the trypsin-like and chymotrypsin-like proteinases were determined to be 25,020, 25,070, 25,060. and 26,260 Da for TL I, TL II, TL III, and CL, respectively. The masses of the CPA enzymes are likely 23,170 and 23,260 Da. whereas the CPB enzyme masses likely are 33,730 and 33,900 Da, The degradation efficiency and cleavage pattern of the trypsin-like proteinases were studied with native myoglobin as a model substrate using CE, MALDI-TOF-MS, and nanoelectrospray mass spectrometry (nESI-MS). The degradation efficiency of the trypsin-like proteinases was found to be approximately 12 and 60 times higher compared to bovine trypsin at 37 degreesC and 1-3 degreesC, respectively. All three fractions of trypsin-like proteinases showed a carboxypeptidase activity in combination with their trypsin activity.

  • 47. Sjodahl, Johan
    et al.
    Lindberg, Peter
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Separation of oligonucleotides in N-methyl-formamide-based polymer matrices by capillary electrophoresis2007In: Journal of Separation Science, ISSN 1615-9306, E-ISSN 1615-9314, Vol. 30, no 1, p. 104-109Article in journal (Refereed)
    Abstract [en]

    N-Methylformamide (NMF)-based matrices for capillary electrophoretic separation of nucleic acids have been developed. The use of an organic solvent as liquid base for the separation matrices allowed a hydrophobic polymer, C-16-derivatized 2-hydroxyethyl cellulose (HEC), to be employed as structural element in the sieving medium. With a matrix consisting of 5% w/v of this polymer dissolved in NMF containing 50 mM ammonium acetate, p(dA)(12-18) and p(dA)(40-60) oligonucleoticles were baseline separated. The addition of ammonium acetate to the buffer and separation matrix resulted in enhanced separation efficiency. Furthermore, it was possible to tailor the sieving performance of the separation medium by the use of a binary mixture of C16-derivatized HEC and PVP. Differences in sieving behavior of the various matrices evaluated are discussed.

  • 48. Sjödahl, J.
    et al.
    Melin, J.
    Griss, P.
    Emmer, Åsa
    KTH, Superseded Departments, Chemistry.
    Stemme, Göran
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Roeraade, Johan
    KTH, Superseded Departments, Chemistry.
    Characterization of micromachined hollow tips for two-dimensional nanoelectrospray mass spectrometry2003In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 17, no 4, p. 337-341Article in journal (Refereed)
    Abstract [en]

    In this work an improved design of chip-based nanoelectrospray nozzles is reported. Two-dimensional matrices of out-of-plane 10 mum i.d. silicon dioxide tips with a tapered shape were manufactured using deep reactive ion etching technology. Using a peptide sample, six micromachined tips and six commercially pulled silica capillary tips were compared employing an ion trap mass spectrometer. At a flow rate of 100 nL/min, the detectability obtained was approximately the same for the two types of tips. The relative standard deviation of the signal-to-noise ratio for the peptides between six different tips was on average 22% for the micromachined tips and 45% for the pulled capillary tips. The usefulness of the micromachined tips for analysis of non-covalent protein-ligand complexes was demonstrated by the analysis of a sample of RNase A and cytidine 2'-monophosphate. In another test, analyzing a tryptic digest of 1 pmol/muL cytochrome C, 18 peptides corresponding to a 82% sequence coverage were detected. Using MS/MS, the whole sequence of an 11 amino acid cytochrome C fragment was obtained. Computer simulations were performed on the shape and magnitude of the electrical field around micromachined and pulled capillary tips. To reach the threshold electric field density at the tip apex required to initiate an electrospray, a higher electrospray voltage was needed for the chip-based tips compared with pulled capillary tips. This is due to the influence of the chip base.

  • 49.
    Sjödahl, Johan
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Kempka, Martin
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Hermansson, Kersti
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Thorsén, Anders
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Chip with twin anchors for reduced ion suppression and improved mass accuracy in MALDI-TOF mass spectrometry2005In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 77, no 3, p. 827-832Article in journal (Refereed)
    Abstract [en]

    A new sample target for matrix-assisted laser desorption/ionization mass spectrometry is described. The target consists of pairs of elevated hydrophilic anchor surfaces, positioned in proximity onto a microchip. The anchors are used to obtain separate preparations of sample and external standard, while both anchor surfaces are irradiated simultaneously by the laser pulse. Using a standard, based on six peptides, a 2-fold improvement in mass accuracy is observed. Also, ion suppression is significantly reduced. With a one peptide calibration standard, 22 tryptic fragments from a BSA digest are detected using the twin-anchor concept, whereas only 14 fragments are detected when the sample and standard are laser-ablated as a mixture from a conventional anchor target. A volume of similar to30 pL of sample solution of angiotensin I is transferred to the anchor surface, under a thin layer of a perfluorocarbon, to prevent a concentration bias due to evaporation. With this arrangement, a detection limit of 1.5 amol was achieved with a signal-to-noise ratio of 22:1.

  • 50.
    Sundqvist, Gustav
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Benkestock, Kurt
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Investigation of multiple binding sites on ribonuclease A using nano-electrospray ionization mass spectrometry2005In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 19, no 8, p. 1011-1016Article in journal (Refereed)
    Abstract [en]

    Multiple non-active site interactions between ribonuclease A (RNAse) and selected target molecules were investigated using nano-electrospray ionization mass spectrometry (nano-ESI-MS). Among the building blocks of RNA, phosphate and ribose showed such multiple interactions. Multiple phosphate interactions survived a high cone voltage, while multiple interactions with D-ribose disappeared already at a low cone voltage. Using nano-ESI-MS, only cytosine among the individual bases appeared to interact with RNAse. Interestingly, guanosine binds to the RNAse surface at high cone voltage, probably as a result of cooperative binding of the sugar and the guanine base. Upon binding of deoxycytidine oligonucleotides with six (dC(6)), nine (dC(9)) and twelve (dC(12)) deoxycytidine nucleotide units to RNAse, the dC(12) Unit showed the strongest interaction. Upon collision-induced dissociation (CID) of the RNAse/dC(6) complex, this complex survived dissociation at an energy level where covalently bound cytosine from dC(6) was lost. This is in contrast to CID of RNAse complexed with mononucleotide cytidine 2'-monophosphate (CMP), which dissociates from the protein without breaking of covalent bonds.

12 1 - 50 of 57
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