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  • 1. Acero Sanchez, Josep Ll.
    et al.
    Joda, Hamdi
    Henry, Olivier Y. F.
    Solnestam, Beata W.
    Kvastad, Linda
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sahlén, Pelin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Laddach, Nadja
    Ramakrishnan, Dheeraj
    Riley, Ian
    Schwind, Carmen
    Latta, Daniel
    O'Sullivan, Ciara K.
    Electrochemical Genetic Profiling of Single Cancer Cells2017In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 89, no 6, p. 3378-3385Article in journal (Refereed)
    Abstract [en]

    Recent understandings in the development and spread of cancer have led to the realization of novel single cell analysis platforms focused on circulating tumor cells (CTCs). A simple, rapid, and inexpensive analytical platform capable of providing genetic information on these rare cells is highly desirable to support clinicians and researchers alike to either support the selection or adjustment of therapy or provide fundamental insights into cell function and cancer progression mechanisms. We report on the genetic profiling of single cancer cells, exploiting a combination of multiplex ligation-dependent probe amplification (MLPA) and electrochemical detection. Cells were isolated using laser capture and lysed, and the mRNA was extracted and transcribed into DNA. Seven markers were amplified by MLPA, which allows for the simultaneous amplification of multiple targets with a single primer pair, using MLPA probes containing unique barcode sequences. Capture probes complementary to each of these barcode sequences were immobilized on a printed circuit board (PCB) manufactured electrode array and exposed to single-stranded MLPA products and subsequently to a single stranded DNA reporter probe bearing a HRP molecule, followed by substrate addition and fast electrochemical pulse amperometric detection. We present asimple, rapid, flexible, and inexpensive approach for the simultaneous quantification of multiple breast cancer related mRNA markers, with single tumor cell sensitivity.

  • 2. Adler, Belinda
    et al.
    Boström, Tove
    KTH, School of Biotechnology (BIO), Proteomics.
    Ekström, Simon
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Laurell, Thomas
    Miniaturized and Automated High-Throughput Verification of Proteins in the ISET Platform with MALDI MS2012In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 84, no 20, p. 8663-8669Article in journal (Refereed)
    Abstract [en]

    A major bottleneck in high-throughput protein production is the validation step, which is why parallel and automated sample processing methods are highly desirable. Also, a miniaturized sample preparation format is preferred, as the reduction of reagent volumes significantly decreases the analysis cost per sample. We have developed an automated and miniaturized protein sequence verification protocol for recombinant proteins utilizing peptide mass fingerprinting and MS/MS analysis. The integrated selective enrichment target (ISET) platform, previously developed in our group, with its dual functionality, being both a sample preparation platform and a MALDI target plate, is employed. All steps including immobilized metal ion affinity chromatography of protein on cobalt-loaded beads, tryptic digestion, and MALDI MS analysis are performed in an array format, without any sample transfers, on the same ISET chip. The automated configuration reduced the sample preparation time significantly. Starting with crude lysate, a full plate of 48 purified, digested samples prepared for MALDI-MS can be generated in 4 h, with only 30 min of operator involvement. This paper demonstrates the utility of the method by parallel analysis of 45 His-tagged human recombinant proteins.

  • 3. Afshar, Majid Ghahraman
    et al.
    Crespo, Gaston A.
    Bakker, Eric
    Coulometric Calcium Pump for Thin Layer Sample Titrations2015In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 87, no 19, p. 10125-10130Article in journal (Refereed)
  • 4. Afshar, Majid Ghahraman
    et al.
    Crespo, Gaston A.
    Bakker, Eric
    Direct Ion Speciation Analysis with Ion-Selective Membranes Operated in a Sequential Potentiometric/Time Resolved Chronopotentiometric Sensing Mode2012In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 84, no 20, p. 8813-8821Article in journal (Refereed)
  • 5. Afshar, Majid Ghahraman
    et al.
    Crespo, Gaston A.
    Bakker, Eric
    Flow Chronopotentiometry with Ion-Selective Membranes for Cation, Anion, and Polyion Detection2016In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 88, no 7, p. 3945-3952Article in journal (Refereed)
  • 6. Afshar, Majid Ghahraman
    et al.
    Crespo, Gaston A.
    Xie, Xiaojiang
    Bakker, Eric
    Direct Alkalinity Detection with Ion-Selective Chronopotentiometry2014In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 86, no 13, p. 6461-6470Article in journal (Refereed)
  • 7. Akter, Farhima
    et al.
    Mie, Masayasu
    Grimm, Sebastian
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Kobatake, Eiry
    Detection of Antigens Using a Protein-DNA Chimera Developed by Enzymatic Covalent Bonding with phiX Gene A2012In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 84, no 11, p. 5040-5046Article in journal (Refereed)
    Abstract [en]

    The chemical reactions used to make antibody DNA conjugates in many immunoassays diminish antigen-binding activity and yield heterogeneous products. Here, we address these issues by developing an antibody-based rolling circle amplification (RCA) strategy using a fusion of phi X174 gene A* protein and Z(mab2s) (A*-Zmab). The phi X174 gene A* protein is an enzyme that can covalently link with DNA, while the Z(mab2s) protein moiety can bind to specific species of antibodies. The DNA in an A*-Zmab conjugate was attached to the A* protein at a site chosen to not interfere with protein function, as determined by enzyme-linked immunosorbent assay (ELISA) and gel mobility shift analysis. The novel A*-Zmab-DNA conjugate retained its binding capabilities to a specific class of murine immunoglobulin gamma 1 (IgG1) but not to rabbit IgG. This indicates the generality of the A*-Zmab-based immuno-RCA assay that can be used in-sandwich ELISA format. Moreover, the enzymatic covalent method dramatically increased the yields of A*-Zmab-DNA conjugates up to 80% after a 15 min reaction. Finally, sensitive detection of human interferon-gamma (IFN-gamma) was achieved by immuno-RCA using our fusion protein in sandwich ELISA format. This new approach of the use of site-specific enzymatic DNA conjugation to proteins should be applicable to fabrication of novel immunoassays for biosensing.

  • 8.
    Araújo, Ana Catarina
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Song, Yajing
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ståhl, Patrik L.
    Brumer, Harry, III
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Activated Paper Surfaces for the Rapid Hybridization of DNA through Capillary Transport2012In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 84, no 7, p. 3311-3317Article in journal (Refereed)
    Abstract [en]

    The development of low-cost, accurate, and equipment-free diagnostic tests is crucial to many clinical, laboratory, and field applications, including forensics and medical diagnostics. Cellulose fiber-based paper is an inexpensive, biodegradable, and renewable resource, the use of which as a biomolecule detection matrix and support confers several advantages compared to traditional materials such as glass. In this context, a new, facile method for the preparation of surface functionalized papers bearing single-stranded probe DNA (ssDNA) for rapid target hybridization via capillary transport is presented. Optimized reaction conditions were developed that allowed the direct, one-step activation of standard laboratory filters by the inexpensive and readily available bifunctional linking reagent, 1,4-phenylenediisothiocyanate. Such papers were thus amenable to subsequent coupling of amine-labeled ssDNA under standard conditions widely used for glass-based supports. The intrinsic wicking ability of the paper matrix facilitated rapid sample elution through arrays of probe DNA, leading to significant, detectable hybridization in the time required for the sample liquid to transit the vertical length of the strip (less than 2 min). The broad applicability of these paper test strips as rapid and specific diagnostics in "real-life" situations was exemplified by the discrimination of amplicons generated from canine and human mitochondrial and genomic DNA in mock forensic samples.

  • 9. Athavale, Rohini
    et al.
    Kokorite, Ilga
    Dinkel, Christian
    Bakker, Eric
    Wehrli, Bernhard
    Crespo, Gaston A.
    Brand, Andreas
    In Situ Ammonium Profiling Using Solid-Contact Ion-Selective Electrodes in Eutrophic Lakes2015In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 87, no 24, p. 11990-11997Article in journal (Refereed)
  • 10.
    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.

  • 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.
    Chmyrov, Andriy
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Sandén, Tor
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Recovery of Photoinduced Reversible Dark States Utilized for Molecular Diffusion Measurements2010In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 82, no 24, p. 9998-10005Article in journal (Refereed)
    Abstract [en]

    For a spatially restricted excitation volume, the effective modulation of the excitation in time is influenced by the passage times of the molecules through the excitation volume. By applying an additional time-modulated excitation, the buildup of photoinduced reversible dark states in fluorescent molecules can be made to vary significantly with their passage times through the excitation volume. The variations in the dark state populations are reflected by the time-averaged fluorescence intensity, which thus can be used to characterize the mobilities of the molecules. The concept was experimentally verified by measuring the fluorescence response of freely diffusing cyanine fluorophores (Cy5), undergoingtrans-cis isomerization when subject to time-modulated excitation in a focused laser beam. From the fluorescence response, and by applying a simple photodynamic model, the transition times of the Cy5 molecules could be well reproduced when applying different laminar flow speeds through the detection volume. The presented approach puts no constraints on sample concentration, no requirements for high time resolution or sensitivity in the detection, nor requires a high fluorescence brightness of the characterized molecules. This can make the concept useful for a broad range of biomolecular mobility studies.

  • 13.
    Chmyrov, Volodymyr
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Spielmann, Thiemo
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Hevekerl, Heike
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Trans-Cis isomerization of lipophilic dyes probing membrane microviscosity in biological membranes and in live cells2015In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 87, no 11, p. 5690-5697Article in journal (Refereed)
    Abstract [en]

    Membrane environment and fluidity can modulate the dynamics and interactions of membrane proteins and can thereby strongly influence the function of cells and organisms in general. In this work, we demonstrate that trans-cis isomerization of lipophilic dyes is a useful parameter to monitor packaging and fluidity of biomembranes. Fluorescence fluctuations, generated by trans-cis isomerization of the thiocarbocyanine dye Merocyanine 540 (MC540), were first analyzed by fluorescence correlation spectroscopy (FCS) in different alcohol solutions. Similar isomerization kinetics of MC540 in lipid vesicles could then also be monitored, and the influence of lipid polarity, membrane curvature, and cholesterol content was investigated. While no influence of membrane curvature and lipid polarity could be observed, a clear decrease in the isomerization rates could be observed with increasing cholesterol contents in the vesicle membranes. Finally, procedures to spatially map photoinduced and thermal isomerization rates on live cells by transient state (TRAST) imaging were established. On the basis of these procedures, MC540 isomerization was studied on live MCF7 cells, and TRAST images of the cells at different temperatures were found to reliably detect differences in the isomerization parameters. Our studies indicate that trans-cis isomerization is a useful parameter for probing membrane dynamics and that the TRAST imaging technique can provide spatial maps of photoinduced isomerization as well as both photoinduced and thermal back-isomerization, resolving differences in local membrane microviscosity in live cells.

  • 14. Crespo, Gaston A.
    et al.
    Afshar, Majid Ghahraman
    Bakker, Eric
    Direct Detection of Acidity, Alkalinity, and pH with Membrane Electrodes2012In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 84, no 23, p. 10165-10169Article in journal (Refereed)
  • 15. Crespo, Gaston A.
    et al.
    Afshar, Majid Ghahraman
    Dorokhin, Denis
    Bakker, Eric
    Thin Layer Coulometry Based on Ion-Exchanger Membranes for Heparin Detection in Undiluted Human Blood2014In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 86, no 3, p. 1357-1360Article in journal (Refereed)
  • 16. Crespo, Gaston A.
    et al.
    Cuartero, Maria
    Bakker, Eric
    Thin Layer Ionophore-Based Membrane for Multianalyte Ion Activity Detection2015In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 87, no 15, p. 7729-7737Article in journal (Refereed)
  • 17. Crespo, Gaston A.
    et al.
    Macho, Santiago
    Bobacka, Johan
    Rius, F. Xavier
    Transduction Mechanism of Carbon Nanotubes in Solid-Contact Ion-Selective Electrodes2009In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 81, no 2, p. 676-681Article in journal (Refereed)
  • 18. Crespo, Gaston A.
    et al.
    Macho, Santiago
    Xavier Rius, F.
    Ion-selective electrodes using carbon nanotubes as ion-to-electron transducers2008In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 80, no 4, p. 1316-1322Article in journal (Refereed)
  • 19. Cuartero, Maria
    et al.
    Acres, Robert G.
    De Marco, Roland
    Bakker, Eric
    Crespo, Gaston A.
    Electrochemical Ion Transfer with Thin Films of Poly(3-octylthiophene)2016In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 88, no 13, p. 6939-6946Article in journal (Refereed)
  • 20. Cuartero, Maria
    et al.
    Crespo, Gaston A.
    Afshar, Majid Ghahraman
    Bakker, Eric
    Exhaustive Thin-Layer Cyclic Voltammetry for Absolute Multianalyte Halide Detection2014In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 86, no 22, p. 11387-11395Article in journal (Refereed)
  • 21. Cuartero, Maria
    et al.
    Crespo, Gaston A.
    Bakker, Eric
    Ionophore-Based Voltammetric Ion Activity Sensing with Thin Layer Membranes2016In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 88, no 3, p. 1654-1660Article in journal (Refereed)
  • 22. Cuartero, Maria
    et al.
    Crespo, Gaston A.
    Bakker, Eric
    Paper-Based Thin-Layer Coulometric Sensor for Halide Determination2015In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 87, no 3, p. 1981-1990Article in journal (Refereed)
  • 23. Cuartero, Maria
    et al.
    Crespo, Gaston A.
    Bakker, Eric
    Polyurethane Ionophore-Based Thin Layer Membranes for Voltammetric Ion Activity Sensing2016In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 88, no 11, p. 5649-5654Article in journal (Refereed)
  • 24. Cuartero, Maria
    et al.
    Crespo, Gaston A.
    Bakker, Eric
    Tandem Electrochemical Desalination-Potentiometric Nitrate Sensing for Seawater Analysis2015In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 87, no 16, p. 8084-8089Article in journal (Refereed)
  • 25.
    Cuartero, Maria
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH). Univ Geneva, Dept Inorgan & Analyt Chem, Quai Ernest Ansermet 30, CH-1211 Geneva, Switzerland.
    Crespo, Gaston
    Univ Geneva, Dept Inorgan & Analyt Chem, Quai Ernest Ansermet 30, CH-1211 Geneva, Switzerland.;KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth, Teknikringen 30, SE-10044 Stockholm, Sweden..
    Cherubini, Thomas
    Univ Geneva, Dept Inorgan & Analyt Chem, Quai Ernest Ansermet 30, CH-1211 Geneva, Switzerland..
    Pankratova, Nadezda
    Univ Geneva, Dept Inorgan & Analyt Chem, Quai Ernest Ansermet 30, CH-1211 Geneva, Switzerland..
    Confalonieri, Fabio
    Idronaut, Via Monte Amiata 10, I-20047 Milan, Italy..
    Massa, Francesco
    Univ Genoa, Dept Earth Environm & Life Sci, Cso Europa 26, I-16132 Genoa, Italy..
    Tercier-Waeber, Mary-Lou
    Univ Geneva, Dept Inorgan & Analyt Chem, Quai Ernest Ansermet 30, CH-1211 Geneva, Switzerland..
    Abdou, Melina
    Univ Bordeaux, UMR CNRS EPOC 5805, Bat 18,Allee Geoffroy St Hilaire, F-33615 Pessac, France..
    Schafer, Jorg
    Univ Bordeaux, UMR CNRS EPOC 5805, Bat 18,Allee Geoffroy St Hilaire, F-33615 Pessac, France..
    Bakker, Eric
    Univ Geneva, Dept Inorgan & Analyt Chem, Quai Ernest Ansermet 30, CH-1211 Geneva, Switzerland..
    In Situ Detection of Macronutrients and Chloride in Seawater by Submersible Electrochemical Sensors2018In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 90, no 7, p. 4702-4710Article in journal (Refereed)
    Abstract [en]

    A new submersible probe for the in situ detection of nitrate, nitrite, and chloride in seawater is presented. Inline coupling of a desalination unit, an acidification unit, and a sensing flow cell containing all-solid-state membrane electrodes allows for the potentiometric detection of nitrate and nitrite after removal of the key interfering ions in seawater, chloride and hydroxide. Thus, the electrodes exhibited attractive analytical performances for the potentiometric detection of nitrate and nitrite in desalinated and acidified seawater: fast response time (t(95) < 12 s), excellent stability (long-term drifts of <0.5 mV h(-1)), good reproducibility (calibration parameter deviation of <3%), and satisfactory accuracy (uncertainties <8%Diff compared to reference technique). The desalination cell, which can be repetitively used for about 30 times, may additionally be used as an exhaustive, and therefore calibration-free, electrochemical sensor for chloride and indirect salinity detection. The detection of these two parameters together with nitrate and nitrite may be useful for the correlation of relative changes in macronutrient levels with salinity cycles, which is of special interest in recessed coastal water bodies. The system is capable of autonomous operation during deployment, with routines for repetitive measurements (every 2 h), data storage and management, and computer visualization of the data in real time. In situ temporal profiles observed in the Arcachon Bay (France) showed valuable environmental information concerning tide-dependent cycles of nitrate and chloride levels in the lagoon, which are here observed for the first time using direct in situ measurements. The submersible probe based on membrane electrodes presented herein may facilitate the study of biogeochemical processes occurring in marine ecosystems by the direct monitoring of nitrate and nitrite levels, which are key chemical targets in coastal waters.

  • 26. 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.

  • 27.
    Du, Zhixue
    et al.
    Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    Yu, Jing
    Shanghai Jiao Tong Univ, Sch Life Sci & Biotechnol, State Key Lab Microbial Metab, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    Li, Fucai
    Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    Deng, Liyun
    Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    Wu, Fang
    Shanghai Jiao Tong Univ, Shanghai Ctr Syst Biomed, Minist Educ, Key Lab Syst Biomed, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    Huang, Xiangyi
    Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    Bergstrand, Jan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Dong, Chaoqing
    Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    Ren, Jicun
    Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    In Situ Monitoring of p53 Protein and MDM2 Protein Interaction in Single Living Cells Using Single-Molecule Fluorescence Spectroscopy2018In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 90, no 10, p. 6144-6151Article in journal (Refereed)
    Abstract [en]

    Protein-protein interactions play a central role in signal transduction, transcription regulations, enzymatic activity, and protein synthesis. The p53 protein is a key transcription factor, and its activity is precisely regulated by the p53-MDM2 interaction. Although the p53-MDM2 interaction has been studied, it is still not clear how p53 structures and external factors influence the p53-MDM2 interaction in living cells. Here, we developed a direct method for monitoring the p53-MDM2 interaction in single living cells using single-molecule fluorescence cross-correlation spectroscopy with a microfluidic chip. First, we labeled p53 and MDM2 proteins with enhanced green fluorescent protein (EGFP) and mCherry, respectively, using lentivirus infection. We then designed various mutants covering the three main domains of p53 (tetramerization, transactivation, and DNA binding domains) and systematically studied effects of p53 protein primary, secondary, and quaternary structures on p53 MDM2 binding affinity in single living cells. We found that p53 dimers and tetramers can bind to MDM2, that the binding affinity of p53 tetramers is higher than that of p53 dimers, and that the affinity is closely correlated to the helicity of the p53 transactivation domain. The hot-spot mutation R175H in the DNA-binding domain reduced the binding of p53 to MDM2. Finally, we studied effects of inhibitors on p53-MDM2 interactions and dissociation dynamics of pS3-MDM2 complexes in single living cells. We found that inhibitors Nutlin 3 alpha and MI773 efficiently inhibited the pS3-MDM2 interaction, but RITA did not work in living cells. This study provides a direct way for quantifying the relationship between protein structure and protein protein interactions and evaluation of inhibitors in living cells.

  • 28.
    Edlund, Ulrica
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Polymer Technology.
    Yu, Yang
    Ryberg, Yingzhi Zhu
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Polymer Technology.
    Krause-Rehberg, Reinhard
    Albertsson, Ann-Christine
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Polymer Technology.
    Positron Lifetime Reveals the Nano Level Packing in Complex Polysaccharide-Rich Hydrolysate Matrixes2012In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 84, no 8, p. 3676-3681Article in journal (Refereed)
    Abstract [en]

    Positron annihilation lifetime spectroscopy (PALS) was used to quantify the free volume and molecular packing in hydrolysate and hemicellulose-based barriers films, derived from process streams during wood processing operations. These hydrolysate films, comprising a fair share of lignin coexisting with poly- and oligo-saccharides, have very low but variable oxygen permeability but differ among themselves with respect to barrier performance as well as molecular weight, degree of branching, and monosaccharide residue main chain composition. From PALS measurements on hydrolysates, the free volume hole radius (r(h)), radius distributions (n(r(h))), volume-weighted hole sizes (<v(h)>(v)), and hole volume distributions (g(v(h))) were calculated showing that the hydrolysate matrixes are very densely packed with small holes. The results show a clear relationship between hydrolysate molecular architecture and composition, the nanolevel molecular packing, and the ability of suppressing the diffusion of oxygen through the film.

  • 29.
    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).

  • 30.
    Elwinger, Fredrik
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Applied Physical Chemistry. GE Healthcare Biosci AB, Bjorkgatan 31, SE-75184 Uppsala, Sweden..
    Wernersson, Jonny
    GE Healthcare Biosci AB, Bjorkgatan 31, SE-75184 Uppsala, Sweden..
    Furo, Istvan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Applied Physical Chemistry. GE Healthcare Biosci AB, Bjorkgatan 31, SE-75184 Uppsala, Sweden..
    Quantifying Size Exclusion by Diffusion NMR: A Versatile Method to Measure Pore Access and Pore Size2018In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 90, no 19, p. 11431-11438Article in journal (Refereed)
    Abstract [en]

    Size-exclusion quantification NMR spectroscopy (SEQNMR) is introduced for measuring equilibrium distribution coefficients, K-eq, in porous media. The porous medium is equilibrated with a polydisperse polymer solution. The original bulk polymer solution and the polymer solution after equilibration but in the absence of the porous medium are analyzed by NMR diffusion experiments. The joint evaluation of the two diffusion attenuation curves under suitable constraints provides the extent by which polymer fractions of particular size were depleted from the solution by pore access. This procedure yields K-eq versus polymer probe size, the selectivity curve that in turn can provide the pore size and its distribution. Simulations probe the performance of the method that is demonstrated experimentally in chromatographic media using dextran polymers. SEQ-NMR and inverse size- exclusion chromatography (ISEC) yield selectivity curves that virtually coincide. Crucial advantages with SEQ-NMR, such as versatility with regard to both the polymer used and porous system explored, high speed, potential for automation, and small required sample volume, are discussed.

  • 31.
    Fazinic, Stjepko
    et al.
    Rudjer Boskovic Inst, Bijenicka 54, Zagreb 10000, Croatia..
    Tadic, Tonic
    Rudjer Boskovic Inst, Bijenicka 54, Zagreb 10000, Croatia..
    Vuksic, Marin
    Rudjer Boskovic Inst, Bijenicka 54, Zagreb 10000, Croatia..
    Rubel, Marek
    KTH, School of Electrical Engineering and Computer Science (EECS), Fusion Plasma Physics.
    Petersson, Per
    KTH, School of Electrical Engineering and Computer Science (EECS), Fusion Plasma Physics.
    Fortuna-Zalesna, Elibieta
    Warsaw Univ Technol, Fac Mat Sci & Technol, Woloska 141, PL-02507 Warsaw, Poland..
    Widdowson, Anna
    Culham Sci Ctr, Culham Ctr Fus Energy, Abingdon OX14 3DB, Oxon, England..
    Ion Microbeam Analyses of Dust Particles and Codeposits from JET with the ITER-Like Wall2018In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 90, no 9, p. 5744-5752Article in journal (Refereed)
    Abstract [en]

    Generation of metal dust in the JET tokamak with the ITER-like wall (ILW) is a topic of vital interest to next-step fusion devices because of safety issues with plasma operation. Simultaneous Nuclear Reaction Analysis (NRA) and Particle Induced X-ray Emission (PIXE) with a focused four MeV He-3 microbeam was used to determine the composition of dust particles related to the JET operation with the ILW. The focus was on "Be-rich particles" collected from the deposition zone on the inner divertor tile. The particles found are composed of a mix of codeposited species up to 120 m in size with a thickness of 30-40 mu m, The main constituents are D from the fusion fuel, Be and W from the main plasma-facing components, and Ni and Cr from the Inconel grills of the antennas for auxiliary plasma heating. Elemental concentrations were estimated by iterative NRA-PIXE analysis. Two types of dust particles were found: (i) larger Be-rich particles with Be concentrations above 90 at% with a deuterium presence of up to 3.4 at% and containing Ni (1-3 at%), Cr (0.4-0.8 at%), W (0.2-0.9 at%), Fe (0.3-0.6 at%), and Cu and Ti in lower concentrations and (ii) small particles rich in Al and/or Si that were in some cases accompanied by other elements, such as Fe, Cu, or Ti or W and Mo.

  • 32. Fornell, Anna
    et al.
    Nilsson, Johan
    Jonsson, Linus
    Rajeswari, Prem Kumar Periyannan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Jönsson, Håkan N.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Tenje, Maria
    Controlled Lateral Positioning of Microparticles Inside Droplets Using Acoustophoresis2015In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 87, no 20, p. 10521-10526Article in journal (Refereed)
    Abstract [en]

    In this paper, we utilize bulk acoustic waves to control the position of micropartides inside droplets in two-phase microfluidic systems and demonstrate a method to enrich the micropartides. In droplet microfluidics, different unit operations are combined and integrated on-chip to miniaturize complex biochemical assays. We present a droplet unit operation capable of controlling the position of micropartides during a trident shaped droplet split. An acoustic standing wave field is generated in the microchannel, and the acoustic forces direct the encapsulated micropartides to the center of the droplets. The method is generic, requires no labeling of the micropartides, and is operated in a noncontact fashion. It was possible to achieve 2+-fold enrichment of polystyrene beads (5 mu m in diameter) in the center daughter droplet with an average recovery of 89% of the beads. Red blood cells were also successfully manipulated inside droplets. These results show the possibility to use acoustophoresis in two-phase systems to enrich micropartides and open up the possibility for new droplet-based assays that are not performed today.

  • 33. Grygolowicz-Pawlak, Ewa
    et al.
    Crespo, Gaston A.
    Afshar, Majid Ghahraman
    Mistlberger, Guenter
    Bakker, Eric
    Potentiometric Sensors with Ion-Exchange Donnan Exclusion Membranes2013In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 85, no 13, p. 6208-6212Article in journal (Refereed)
    Abstract [en]

    Potentiometric sensors that exhibit a non-Hofmeister selectivity sequence are normally designed by selective chemical recognition elements in the membrane. In other situations, when used as detectors in separation science, for example, membranes that respond equally to most ions are preferred. With so-called liquid membranes, a low selectivity is difficult to accomplish since these membranes are intrinsically responsive to lipophilic species. Instead, the high solubility of sample lipids in an ionophore-free sensing matrix results in a deterioration of the response. We explore here potentiometric sensors on the basis of ion-exchange membranes commonly used in fuel cell applications and electrodialysis, which have so far not found their way into the field of ion-selective electrodes. These membranes act as Donnan exclusion membranes as the ions are not stripped of their hydration shell as they interact with the membrane. Because of this, lipophilic ions are no longer preferred over hydrophilic ones, making them promising candidates for the detection of abundant ions in the presence of lipophilic ones or as detectors in separation science. Two types of cation-exchanger membranes and one anion-exchange membrane were characterized, and potentiometric measuring ranges were found to be Nernstian over a wide range down to about 10 μM concentrations. Depending on the specific membrane, lipophilic ions gave equal response to hydrophilic ones or were even somewhat discriminated. The medium and long-term stability and reproducibility of the electrode signals were found to be promising when evaluated in synthetic and whole blood samples.

  • 34.
    Gutierrez Arenas, Omar
    et al.
    Universidad de la Habana; Department of Biochemistry, Biomedical Center, Uppsala University.
    Chavez, M.
    Lissi, E.
    A Theoretical Approach to Some Analytical Properties of Heterogeneous Enzymatic Assays2004In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 76, no 9, p. 2664-2668Article in journal (Refereed)
    Abstract [en]

    Heterogeneous enzymatic assays (HEA), where an enzyme in solution acts upon an immobilized substrate, are been increasingly used. Given their high throughput and versatility they hold great potential for developing massive enzyme inhibitor screening. However, current HEA lack, in general, rigorous quantitative use. This is in part due to technical problems as a multiplicity of suboptimal substrate populations achieved with traditional immobilization techniques but, more importantly, is due to a poor understanding of the particular kinetic behavior of these systems. This paper addresses the kinetic features of HEA that arise from the very low amount of solid-phase substrate and the resulting inalterability of the free enzyme concentration during the assay, which classify HEA as enzyme quasi-saturable systems (EQSS). We assessed the optimal enzyme concentration working range and time of reaction. We also considered certain attributes of HEA for evaluating isosteric inhibitors. These studies were done on the basis of a simplified model for the kinetics of EQSS and a formal splitting of the functional factor of the analytical sensitivity of an enzymatic assay into [E-0]/K-m-dependent and temporal components.

  • 35. Guzinski, M.
    et al.
    Jarvis, J. M.
    Perez, F.
    Pendley, B. D.
    Lindner, E.
    De Marco, R.
    Crespo, Gaston A.
    Acres, R. G.
    Walker, R.
    Bishop, J.
    PEDOT(PSS) as Solid Contact for Ion-Selective Electrodes: The Influence of the PEDOT(PSS) Film Thickness on the Equilibration Times2017In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 89, no 6, p. 3508-3516Article in journal (Refereed)
    Abstract [en]

    To understand the rate determining processes during the equilibration of poly(3,4-ethylenedioxythiophene):polystyrenesulfonate-based (PEDOT(PSS)-based) solid contact (SC) ion-selective electrodes (ISEs), the surfaces of Pt, Au, and GC electrodes were coated with 0.1, 1.0, 2.0, and 4.0 μm thick galvanostatically deposited PEDOT(PSS) films. Next, potential vs time transients were recorded with these electrodes, with and without an additional potassium ion-selective membrane (ISM) coating, following their first contact with 0.1 M KCl solutions. The transients were significantly different when the multilayered sensor structures were assembled on Au or GC compared to Pt. The differences in the rate of equilibration were interpreted as a consequence of differences in the hydrophilicity of PEDOT(PSS) in contact with the substrate electrode surfaces based on X-ray photoelectron spectroscopy (XPS) and synchrotron radiation-XPS (SR-XPS) analysis of 10–100 nm thick PEDOT(PSS) films. The influence of the layer thickness of the electrochemically deposited PEDOT(PSS)-films on the hydrophilicity of these films has been documented by contact angle measurements over PEDOT(PSS)-coated Au, GC, and Pt electrode surfaces. This study demonstrates that it is possible to minimize the equilibration (conditioning) time of SC ISEs with aqueous solutions before usage by optimizing the thickness of the SC layer with a controlled ISM thickness. PEDOT(PSS)-coated Au and GC electrodes exhibit a significant negative potential drift during their equilibration in an aqueous solution. By coating the PEDOT(PSS) surface with an ISM, the negative potential drift is compensated by a positive potential drift related to the hydration of the ISM and activity changes at the PEDOT(PSS)|ISM interface. The potential drifts related to activity changes in the ISM have been determined by a novel adaptation of the “sandwich membrane” method.

  • 36. Gösch, Michael
    et al.
    Blom, Hans
    KTH, Superseded Departments, Microelectronics and Information Technology, IMIT.
    Holm, Johan
    Heino, Toni
    Rigler, Rudolf
    Hydrodynamic Flow Profiling in Microchannel Structures by Single Molecule Fluorescence Correlation Spectroscopy2000In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 72, no 14, p. 3260-3265Article in journal (Refereed)
    Abstract [en]

    In this paper we demonstrate high spatial resolution hydrodynamic flow profiling in silicon wafer based microchannels using single molecule fluorescence correlation spectroscopy (FCS). We have used confocal fluorescence microscopy to detect single tetramethylrhodamine (TMR-4-dUTP) biomolecules traversing a l fL volume element defined by an argon laser beam focus. By elevating a (10-10 M) reservoir of diluted analyte, a continuous hydrodynamic flow through the microstructure could be accomplished. The microchannel was then scanned with a diffraction-limited focus in 1-μm steps in both the vertical and the horizontal directions to determine the flow profile across a 50 × 50 μm2 channel. The flow profile measured was parabolic in both dimensions, thereby showing a Poiseuille laminar flow profile. Future microstructures can hereby be nondestructively investigated with the use of high spatial resolution confocal correlation microscopy.

  • 37. 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.

  • 38.
    Hauser, Janosch
    et al.
    KTH, School of Electrical Engineering and Computer Science (EECS), Micro and Nanosystems.
    Lenk, Gabriel
    KTH, School of Electrical Engineering and Computer Science (EECS), Micro and Nanosystems.
    Hansson, Jonas
    KTH, School of Electrical Engineering and Computer Science (EECS), Micro and Nanosystems.
    Beck, Olof
    Karolinska Inst, Dept Lab Med, S-14186 Stockholm, Sweden..
    Stemme, Göran
    KTH, School of Electrical Engineering and Computer Science (EECS), Micro and Nanosystems.
    Roxhed, Niclas
    KTH, School of Electrical Engineering and Computer Science (EECS), Micro and Nanosystems.
    High-Yield Passive Plasma Filtration from Human Finger Prick Blood2018In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 90, no 22, p. 13393-13399Article in journal (Refereed)
    Abstract [en]

    Whole-blood microsampling provides many benefits such as remote, patient-centric, and minimally invasive sampling. However, blood plasma, and not whole blood, is the prevailing matrix in clinical laboratory investigations. The challenge with plasma microsampling is to extract plasma volumes large enough to reliably detect low-concentration analytes from a small finger prick sample. Here we introduce a passive plasma filtration device that provides a high extraction yield of 65%, filtering 18 mu L of plasma from 50 mu L of undiluted human whole blood (hematocrit 45%) within less than 10 min. The enabling design element is a wedge-shaped connection between the blood filter and the hydrophilic bottom surface of a capillary channel. Using finger prick and venous blood samples from more than 10 healthy volunteers, we examined the filtration kinetics of the device over a hematocrit range of 35-55% and showed that 73 +/- 8% of the total protein content was successfully recovered after filtration. The presented plasma filtration device tackles a major challenge toward patient-centric blood microsampling by providing high-yield plasma filtration, potentially allowing reliable detection of low-concentration analytes from a blood microsample.

  • 39.
    Hauser, Janosch
    et al.
    KTH, School of Electrical Engineering and Computer Science (EECS), Micro and Nanosystems.
    Lenk, Gabriel
    KTH, School of Electrical Engineering and Computer Science (EECS), Micro and Nanosystems.
    Ullah, Shahid
    Karolinska Univ Hosp, Clin Pharmacol, S-11486 Stockholm, Sweden..
    Beck, Olof
    Karolinska Univ Hosp, Clin Pharmacol, S-11486 Stockholm, Sweden..
    Stemme, Göran
    KTH, School of Electrical Engineering and Computer Science (EECS), Micro and Nanosystems.
    Roxhed, Niclas
    KTH, School of Electrical Engineering and Computer Science (EECS), Micro and Nanosystems.
    An Autonomous Microfluidic Device for Generating Volume-Defined Dried Plasma Spots2019In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 91, no 11, p. 7125-7130Article in journal (Refereed)
    Abstract [en]

    Obtaining plasma from a blood sample and preparing it for subsequent analysis is currently a laborious process involving experienced health-care professionals and centrifugation. We circumvent this by utilizing capillary forces and microfluidic engineering to develop an autonomous plasma sampling device that filters and stores an exact amount of plasma as a dried plasma spot (DPS) from a whole blood sample in less than 6 min. We tested 24 prototype devices with whole blood from 10 volunteers, various input volumes (40-80 mu L), and different hematocrit levels (39-45%). The resulting mean plasma volume, assessed gravimetrically, was 11.6 mu L with a relative standard deviation similar to manual pipetting (3.0% vs 1.4%). LC-MS/MS analysis of caffeine concentrations in the generated DPS (12 duplicates) showed a strong correlation (R-2 = 0.99) to, but no equivalence with, concentrations prepared from corresponding plasma obtained by centrifugation. The presented autonomous DPS device may enable patient-centric plasma sampling through minimally invasive finger-pricking and allow generatation of volume-defined DPS for quantitative blood analysis.

  • 40. Jansod, Sutida
    et al.
    Afshar, Majid Ghahraman
    Crespo, Gaston A.
    Bakker, Eric
    Alkalinization of Thin Layer Samples with a Selective Proton Sink Membrane Electrode for Detecting Carbonate by Carbonate-Selective Electrodes2016In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 88, no 7, p. 3444-3448Article in journal (Refereed)
    Abstract [en]

    Potentiometry is known to be sensitive to so-called free ion activity and is a potentially valuable tool in environmental speciation analysis. Here, the direct detection of free and total carbonate is demonstrated by alkalinization of a thin layer sample (∼100 μm), which is electrochemically triggered at a pH responsive membrane placed opposite a carbonate-selective membrane electrode. The concept may serve as a promising future methodology for in situ environmental sensing applications where traditional sampling and pretreatment steps are no longer required. The possibility of increasing the pH of the sample was demonstrated first with a proton selective membrane (pH readout at zero current) placed opposite the thin layer gap. An optimal applied potential (600 mV) for 300 s resulted in a pH increase of 4 units in an artificial sample, with a relative standard deviation (RSD) of ∼2%. The pH probe was subsequently replaced by a solid contact carbonate selective electrode for the determination of carbonate species (4.17 μM) in a sample of 1 mM NaHCO3. Increasing the pH to 12.1 by the electrochemically controlled proton sink allowed one to convert bicarbonate to the detectable carbonate species. Initial bicarbonate concentration (∼1 mM) was obtained as the difference between the converted bicarbonate and the initial carbonate concentration. An initial application of this concept was illustrated by the speciation analysis of an unfiltered sample from the Arve river (12.3 ± 0.2 μM and 22.5 ± 0.3 mM carbonate and bicarbonate, respectively). The values were confirmed by volumetric titration.

  • 41.
    Jansod, Sutida
    et al.
    Univ Geneva, Dept Inorgan & Analyt Chem, Quai Ernest Ansermet 30, CH-1211 Geneva 4, Switzerland..
    Cuartero, Maria
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH). Univ Geneva, Dept Inorgan & Analyt Chem, Quai Ernest Ansermet 30, CH-1211 Geneva 4, Switzerland.;KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth, Stockholm, Sweden..
    Cherubini, Thomas
    Univ Geneva, Dept Inorgan & Analyt Chem, Quai Ernest Ansermet 30, CH-1211 Geneva 4, Switzerland..
    Bakker, Eric
    Univ Geneva, Dept Inorgan & Analyt Chem, Quai Ernest Ansermet 30, CH-1211 Geneva 4, Switzerland..
    Colorimetric Readout for Potentiometric Sensors with Closed Bipolar Electrodes2018In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 90, no 11, p. 6376-6379Article in journal (Refereed)
    Abstract [en]

    We present here a general strategy to translate potential change at a potentiometric probe into a tunable color readout. It is achieved with a closed bipolar electrode where the ion-selective component is confined to one end of the electrode while color is generated at the opposite pole, allowing one to physically separate the detection compartment from the sample. An electrical potential is imposed across the bipolar electrode by solution contact such that the potentiometric signal change at the sample side modulates the potential at the detection side. This triggers the turnover of a redox indicator in the thin detection layer until a new equilibrium state is established. The approach is demonstrated in separate experiments with a chloride responsive Ag/AgCl element and a liquid membrane based calcium-selective membrane electrode, using the redox indicator ferroin in the detection compartment. The principle can be readily extended to other ion detection materials and optical readout principles.

  • 42. Jarolimova, Zdenka
    et al.
    Crespo, Gaston A.
    Xie, Xiaojiang
    Afshar, Majid Ghahraman
    Pawlak, Marcin
    Bakker, Eric
    Chronopotentiometric Carbonate Detection with All-Solid-State lonophore-Based Electrodes2014In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 86, no 13, p. 6307-6314Article in journal (Refereed)
    Abstract [en]

    We present here for the first time an all-solid-state chronopotentiometric ion sensing system based on selective ionophores, specifically for the carbonate anion. A chronopotentiometric readout is attractive because it may allow one to obtain complementary information on the sample speciation compared to zero-current potentiometry and detect the sum of labile carbonate species instead of only ion activity. Ferrocene covalently attached to the PVC polymeric chain acts as an ion-to-electron transducer and provides the driving force to initiate the sensing process at the membrane–sample interface. The incorporation of a selective ionophore for carbonate allows one to determine this anion in a background electrolyte. Various inner electrolyte and all-solid-state-membrane configurations are explored, and localized carbonate depletion is only observed for systems that do not contain ion-exchanger additives. The square root of the transition times extracted from the inflection point of the chronopotentiograms as a function of carbonate specie concentration follows a linear relationship. The observed linear range is 0.03–0.35 mM in a pH range of 9.50–10.05. By applying the Sand equation, the diffusion coefficient of carbonate is calculated as (9.03 ± 0.91) 10–6 cm2 s–1, which corresponds to the established value. The reproducibility of assessed carbonate is better than 1%. Additionally, carbonate is monitored during titrimetric analysis as a precursor to an in situ environmental determination. Based on these results, Fc-PVC membranes doped with ionophores may form the basis of a new family of passive/active all-solid-state ion selective electrodes interrogated by a current pulse.

  • 43.
    Kong, Na
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Zhou, Juan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Park, JaeHyeung
    University of Massachusetts, United States .
    Xie, Sheng
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Ramström, Olof
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Yan, Mingdi
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry. University of Massachusetts, United States .
    Quantitative Fluorine NMR To Determine Carbohydrate Density on Glyconanomaterials Synthesized from Perfluorophenyl Azide-Functionalized Silica Nanoparticles by Click Reaction2015In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 87, no 18, p. 9451-9458Article in journal (Refereed)
    Abstract [en]

    A quantitative fluorine NMR (F-19 qNMR) method was developed to determine the carbohydrate density on glyconanomaterials. Mannose (Man)- and galactose (Gal)-conjugated silica nanoparticles (SNPs) were synthesized from perfluorophenyl azide (PFPA)-functionalized SNPs and propargylated Man or Gal by copper-catalyzed azide-alkyne cycloaddition (click reaction). After treating PFPA-SNPs or Man-SNPs with hydrofluoric acid followed by lyophilization, the remaining residues were directly subjected to F-19 NMR analysis. The density of PFPA on PFPA-SNP was determined to be 7.7 +/- 0.2 x 10(-16) nmol/nm(2) and Man on Man-SNP to be 6.4 +/- 0.2 x 10(-16) nmol/nm(2) giving a yield of similar to 83% for the click coupling reaction. The apparent dissociation constant (K-d) of Man-SNPs with fluorescein isothiocyanate(FITC)-concanavalin A (Con A) was determined using a fluorescence competition assay to be 0.289 +/- 0.003 mu M, which represents more than 3 orders of magnitude affinity increase compared to free Man with Con A.

  • 44.
    Krmpot, Aleksandar J.
    et al.
    Karolinska Inst, CMM, Dept Clin Neurosci CNS, S-17176 Stockholm, Sweden.;Univ Belgrade, Inst Phys Belgrade, Belgrade 11080, Serbia..
    Nikolic, Stanko N.
    Karolinska Inst, CMM, Dept Clin Neurosci CNS, S-17176 Stockholm, Sweden.;Univ Belgrade, Inst Phys Belgrade, Belgrade 11080, Serbia..
    Oasa, Sho
    Karolinska Inst, CMM, Dept Clin Neurosci CNS, S-17176 Stockholm, Sweden..
    Papadopoulos, Dimitrios K.
    Max Planck Inst Mol Cell Biol & Genet, D-01307 Dresden, Germany.;Univ Edinburgh, Inst Genet & Mol Med, MRC Human Genet Unit, Edinburgh EH4 2XU, Midlothian, Scotland..
    Vitali, Marco
    Sicoya GmbH, D-12489 Berlin, Germany..
    Oura, Makoto
    Hokkaido Univ, Fac Adv Life Sci, Lab Mol Cell Dynam, Sapporo, Hokkaido 0010021, Japan..
    Mikuni, Shintaro
    Hokkaido Univ, Fac Adv Life Sci, Lab Mol Cell Dynam, Sapporo, Hokkaido 0010021, Japan..
    Thyberg, Per
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Tisa, Simone
    MPD, I-39100 Bolzano, Italy..
    Kinjo, Masataka
    Hokkaido Univ, Fac Adv Life Sci, Lab Mol Cell Dynam, Sapporo, Hokkaido 0010021, Japan..
    Nilsson, Lennart
    Karolinska Inst, Dept Biosci & Nutr, S-14183 Huddinge, Sweden..
    Terenius, Lars
    Karolinska Inst, CMM, Dept Clin Neurosci CNS, S-17176 Stockholm, Sweden..
    Rigler, Rudolf
    Karolinska Inst, CMM, Dept Clin Neurosci CNS, S-17176 Stockholm, Sweden.;Karolinska Inst, Dept Med Biochem & Biophys MBB, S-17177 Stockholm, Sweden..
    Vukojevic, Vladana
    Karolinska Inst, CMM, Dept Clin Neurosci CNS, S-17176 Stockholm, Sweden..
    Functional Fluorescence Microscopy Imaging: Quantitative Scanning-Free Confocal Fluorescence Microscopy for the Characterization of Fast Dynamic Processes in Live Cells2019In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 91, no 17, p. 11129-11137Article in journal (Refereed)
    Abstract [en]

    Functional fluorescence microscopy imaging (fFMI), a time-resolved (21 mu s/frame) confocal fluorescence microscopy imaging technique without scanning, is developed for quantitative characterization of fast reaction-transport processes in solution and in live cells. The method is based on massively parallel fluorescence correlation spectroscopy (FCS). Simultaneous excitation of fluorescent molecules in multiple spots in the focal plane is achieved using a diffractive optical element (DOE). Fluorescence from the DOE-generated 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector comprising 32 x 32 single-photon avalanche photodiodes (SPADs). Software for data acquisition and fast auto- and cross-correlation analysis by parallel signal processing using a graphic processing unit (GPU) allows temporal autocorrelation across all pixels in the image frame in 4 s and cross-correlation between first- and second-order neighbor pixels in 45 s. We present here this quantitative, time-resolved imaging method with single-molecule sensitivity and demonstrate its usefulness for mapping in live cell location-specific differences in the concentration and translational diffusion of molecules in different subcellular compartments. In particular, we show that molecules without a specific biological function, e.g., the enhanced green fluorescent protein (eGFP), exhibit uniform diffusion. In contrast, molecules that perform specialized biological functions and bind specifically to their molecular targets show location-specific differences in their concentration and diffusion, exemplified here for two transcription factor molecules, the glucocorticoid receptor (GR) before and after nuclear translocation and the Sex combs reduced (Scr) transcription factor in the salivary gland of Drosophila ex vivo.

  • 45. Lan, Wen-Jie
    et al.
    Zou, Xu U.
    Hamedi, Mahiar
    Hu, Jinbo
    Parolo, Claudio
    Maxwell, E. Jane
    Bühlmann, Philippe
    Whitesides, George M.
    Paper-Based Potentiometric Ion Sensing2014In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 86, no 19, p. 9548-9553Article in journal (Refereed)
    Abstract [en]

    This paper describes the design and fabrication of ion-sensing electrochemical paper-based analytical devices (EPADs) in which a miniaturized paper reference electrode is integrated with a small ion-selective paper electrode (ISPE) for potentiometric measurements. Ion-sensing EPADs use printed wax barriers to define electrochemical sample and reference zones. Single-layer EPADs for sensing of chloride ions include wax-defined sample and reference zones that each incorporate a Ag/AgCl electrode. In EPADs developed for other electrolytes (potassium, sodium, and calcium ions), a PVC-based ion-selective membrane is added to separate the sample zone from a paper indicator electrode. After the addition of a small volume (less than 10 μL) of sample and reference solutions to different zones, ion-sensing EPADs exhibit a linear response, over 3 orders of magnitude, in ranges of electrolyte concentrations that are relevant to a variety of applications, with a slope close to the theoretical value (59.2/z mV). Ion-selective EPADs provide a portable, inexpensive, and disposable way of measuring concentrations of electrolyte ions in aqueous solutions.

  • 46.
    Laurell, Fredrik
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Laser Physics.
    Jansson, M.
    Roeraade, J.
    Laser-inducedfluorescence detection in capillary electrophoresis with blue light from afrequency-doubled diode laser1993In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882Article in journal (Refereed)
  • 47.
    Lenk, Gabriel
    et al.
    KTH, School of Electrical Engineering and Computer Science (EECS), Micro and Nanosystems.
    Ullah, S.
    Stemme, Göran
    KTH, School of Electrical Engineering and Computer Science (EECS), Micro and Nanosystems.
    Beck, O.
    Roxhed, Niclas
    KTH, School of Electrical Engineering and Computer Science (EECS), Micro and Nanosystems.
    Evaluation of a Volumetric Dried Blood Spot Card Using a Gravimetric Method and a Bioanalytical Method with Capillary Blood from 44 Volunteers2019In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 91, no 9, p. 5558-5565Article in journal (Refereed)
    Abstract [en]

    Dried blood spot (DBS) sampling is a promising method for collection of microliter blood samples. However, hematocrit-related bias in combination with subpunch analysis can result in inaccurate quantification of analytes in DBS samples. In this study we use a microfluidic DBS card, designed to automatically collect fixed volume DBS samples irrespective of the blood hematocrit, to measure caffeine concentration in normal finger prick samples obtained from 44 human individuals. Caffeine levels originating from blood drops of unknown volume collected on the volumetric microfluidic DBS card were compared to volume-controlled pipetted DBS samples from the same finger prick. Hematocrit independence and volumetric sampling performances were also verified on caffeine-spiked blood samples in vitro, using both LC-MS/MS and gravimetric methods, on hematocrits from 26 to 62%. The gravimetric measurements show an excellent metering performance of the microfluidic DBS card, with a mean blood sample volume of 14.25 μL ± 3.0% (n = 51). A measured mean bias below 2.9% compared to normal hematocrit (47%) demonstrates that there is no significant hematocrit-induced bias. LC-MS/MS measurements confirm low CV and hematocrit independence of the sampling system and exhibit no substantial mean bias compared to pipetted DBS. Tests with 44 individuals demonstrated applicability of the microfluidic DBS card for direct finger prick blood sampling, and measured caffeine concentrations show a good agreement with measurements of pipetted DBS. The presented concept demonstrates a good volumetric performance which can help to improve the accuracy of DBS analysis by analyzing a whole spot, equivalent to a defined volume of liquid blood.

  • 48.
    Martín-Yerga, Daniel
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemical Engineering, Applied Electrochemistry. DropSens, Soc Limitada, Edificio CEEI,Parque Tecnol Asturias, Llanera 33428, Asturias, Spain.
    Perez-Junquera, Alejandro
    DropSens, Soc Limitada, Edificio CEEI,Parque Tecnol Asturias, Llanera 33428, Asturias, Spain..
    Begona Gonzalez-Garcia, Maria
    DropSens, Soc Limitada, Edificio CEEI,Parque Tecnol Asturias, Llanera 33428, Asturias, Spain..
    Hernandez-Santos, David
    DropSens, Soc Limitada, Edificio CEEI,Parque Tecnol Asturias, Llanera 33428, Asturias, Spain..
    Fanjul-Bolado, Pablo
    DropSens, Soc Limitada, Edificio CEEI,Parque Tecnol Asturias, Llanera 33428, Asturias, Spain..
    In Situ Spectroelectrochemical Monitoring of Dye Bleaching after Electrogeneration of Chlorine-Based Species: Application to Chloride Detection2018In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 90, no 12, p. 7442-7449Article in journal (Refereed)
    Abstract [en]

    Spectroelectrochemical techniques are becoming increasingly versatile tools to solve a diverse range of analytical problems. Herein, the use of in situ real-time luminescence spectroelectrochemistry to quantify chloride ions is demonstrated. Utilizing the bleaching effect of chlorine-based electrogenerated products after chloride oxidation, it is shown that the fluorescence of the rhodamine 6G dye decreases proportionally to the initial chloride concentration in solution. A strong decrease of fluorescence is observed in acidic media compared to a lower decrease in alkaline media, which suggests that Cl-2, favorably generated at low pH, could be the main species responsible for the fluorescence loss. This fact is corroborated with chronoamperometric measurements where the complete loss of fluorescence for the bulk solution is achieved. A fast mass transfer is needed to explain this behavior, in agreement with the generation of gaseous species such as Cl-2. Chloride detection was performed in artificial sweat samples in less than 30 s with great accuracy. This electrochemical/optical combined approach allows us to quantify species that are difficult to measure by electrochemistry due to the inadequate resolution of their redox processes or being without significant optical properties.

  • 49. Melander, C.
    et al.
    Momcilovic, Dane
    Nilsson, C.
    Bengtsson, M.
    Schagerlof, H.
    Tjerneld, F.
    Laurell, T.
    Reimann, C. T.
    Gorton, L.
    Microchip immobilized enzyme reactors for hydrolysis of methyl cellulose2005In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 77, no 10, p. 3284-3291Article in journal (Refereed)
    Abstract [en]

    Microchip immobilized enzyme reactors (mu IMERs) with immobilized endoglucanases were applied for the hydrolysis of methyl cellulose (MC). MCs of various molecular weights were hydrolyzed using two mu IMERs containing immobilized celloendoglucanase Cel 5A from Bacillus agaradhaerens (BaCel 5A) connected in series. Hydrolysis by the mu IMER could be confirmed from the average molar masses and molar mass distributions measured by size exclusion chromatography (SEC) with online multiangle light scattering and refractive index detection. Methylated cellooligosaccharides with degrees of polymerization (DP) between 1 and 6 formed during hydrolysis were analyzed by direct infusion electrospray ionization ion-trap mass spectrometry (ESI-ITMS). Mass spectra of mu IMER- and batch-hydrolyzed samples were compared and no significant differences were found, indicating that mu IMER hydrolysis was as efficient as conventional batch hydrolysis. A fast and automated hydrolysis with online MS detection was achieved by connecting the mu IMER to high-performance liquid chromatography and ESI-ITMS. This online separation reduced the relative intensities of interfering signals and increased the signal-to-noise ratios in MS. The mu IMER hydrolysates were also subjected to SEC interfaced with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. With this technique, oligomers with DP 3-30 could be detected. The hydrolysis by they mu-IMER was performed within 60 min, i.e. significantly faster compared with batch hydrolysis usually performed for at least 24 h. The mu IMER also allowed hydrolysis after 10 days of continuous use. The method presented in this work offers new approaches for the analysis of derivatized cellulose and provides the possibility of convenient online, fast, and more versatile analysis compared with the traditional batch method.

  • 50.
    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.

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