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  • 101.
    Russom, Aman
    et al.
    KTH, School of Electrical Engineering (EES), Microsystem Technology (Changed name 20121201).
    Haasl, Sjoerd
    KTH, School of Electrical Engineering (EES), Microsystem Technology (Changed name 20121201).
    Brookes, Anthony J.
    Andersson, Helene
    KTH, School of Electrical Engineering (EES), Microsystem Technology (Changed name 20121201).
    Stemme, Göran
    KTH, School of Electrical Engineering (EES), Microsystem Technology (Changed name 20121201).
    Ultra-rapid melting curve analysis on beads for high-throughput genotyping of single nucleotide polymorphism2005In: Micro Total Analysis Systems - Proceedings of MicroTAS 2005 Conference: 9th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Transducers Research Foundations, 2005, p. 1006-1008Conference paper (Refereed)
    Abstract [en]

    This report describes a rapid solid-phase melting curve analysis method based on dynamic allele-specific hybridization (DASH) for single nucleotide polymorphism (SNP) genotyping. Beads with DNA duplexes are immobilized on the surface of a microheater chip. SNP on PCR products were scored, illustrating the sensitivity and robustness of the system. Single-bead detection and multiplexing were performed with a heating rate more than 20 times faster than conventional DASH. Hence, analyses that took more than 15 minutes could be performed in less than 1 minute, enabling ultra-rapid SNP analysis.

  • 102.
    Russom, Aman
    et al.
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Haasl, Sjoerd
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Ohlander, Anna
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Andersson, Helene
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Stemme, Göran
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Genotyping by dynamic heating of monolayered beads on a microheated surface2004In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 25, no 21-22, p. 3712-3719Article in journal (Refereed)
    Abstract [en]

     A miniaturized bead-based dynamic allele-specific hybridization (DASH) approach for sing le-nucleotide polymorphism analysis is presented. Chips with integrated heater and temperature sensors for open-surface DNA analysis were microfabricated. Microcontact printing using a poly(dimethylsiloxane) (PDMS) stamp was employed to create monolayers of immobilized beads on the surface of the chip. This chip allows fast, well-controllable temperature ramping. The temperature distribution was homogeneous over the entire heater area. All three possible variants of an SNP site of a synthesized oligonucleotide were accurately scored using the bead-based DASH approach. Our assay has a nonoptimized temperature ramping rate of 4degreesC-6degreesC/min compared to earlier reported values of 2degreesC-3degreesC/min, thereby reducing the total analysis time by a factor of 2. Reliable DASH measurement data from areas as small as 12 x 13 mum was achieved. Our bead-based DASH approach has enabled a dramatic volume reduction and is a step towards developing a cost-effective high-throughput DASH method on arrays of single beads.

  • 103.
    Russom, Aman
    et al.
    KTH, School of Electrical Engineering (EES), Microsystem Technology (Changed name 20121201).
    Haasl, Sjoerd
    KTH, School of Electrical Engineering (EES), Microsystem Technology (Changed name 20121201).
    Ohlander, Anna
    KTH, School of Electrical Engineering (EES), Microsystem Technology (Changed name 20121201).
    Mayr, T.
    Karolinska Institute.
    Brookes, A. J.
    Karolinska Institute.
    Andersson, Helene
    KTH, School of Electrical Engineering (EES), Microsystem Technology (Changed name 20121201).
    Stemme, Göran
    KTH, School of Electrical Engineering (EES), Microsystem Technology (Changed name 20121201).
    Genotyping by dynamic heating of monolayered beads on a microheater surface2005In: Micro Total Analysis Systems 2004 / [ed] Laurell T; Nilsson J; Jensen K; Harrison DJ, Royal Society of Chemistry, 2005, no 297, p. 303-305Conference paper (Refereed)
    Abstract [en]

    This paper presents SNP scoring by DASH technology by employing dynamic heating of beads immobilized on a chip with integrated heater and sensor. The microfabricated chip designed for open-surface DNA analysis allows fast, well controllable temperature ramping and homogeneous temperature distribution over the entire heater area. Beads containing DNA duplexes are immobilized on the surface of the chip by microcontact printing using a PDMS stamp. All three possible variants of a SNP site of an oligonucleotide were accurately scored using the bead-based DASH approach. Using the chip, the total analysis time could easily be reduced by a factor 2 compared with the current DASH assay.

  • 104.
    Russom, Aman
    et al.
    KTH, School of Electrical Engineering (EES), Microsystem Technology (Changed name 20121201).
    Tooke, N.
    Andersson, Helene
    KTH, School of Electrical Engineering (EES), Microsystem Technology (Changed name 20121201).
    Stemme, Göran
    KTH, School of Electrical Engineering (EES), Microsystem Technology (Changed name 20121201).
    Pyrosequencing in a microfluidic flow-through device2005In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 77, no 23, p. 7505-7511Article in journal (Refereed)
    Abstract [en]

    To explore genome variation meaningfully, there is a critical need for a high-throughput and inexpensive platform for DNA analysis. Pyrosequencing is a nonelectrophoretic bioluminometric DNA sequencing method that uses a four-enzyme mixture reaction to monitor nucleotide incorporation in real time. Currently, the commercialized pyrosequencing technique is limited to a 96-microtiter plate format. However, high throughput and inexpensive pyrosequencing is required to meet the need of screening large numbers of samples. We present here DNA pyrosequencing on a nanoliter-volume microfluidic platform. The microfluidic approach involves the trapping of the DNA on microbeads in an on-chip filter chamber and flow-through of the pyrosequencing reagents to monitor the reaction in real time. Two single-nucleotide polymorphisms were successfully scored to evaluate the microfluidic platform. In addition to significantly reducing reagent costs, microfluidic systems promise to improve the read length by eliminating intermediate product accumulation by constant removal of unincorporated nucleotides and elimination of dilution effects at each reaction cycle in the current plate format. Although only one filter chamber was used in this study, the platform should be readily adaptable to parallel analyses of nanoliter samples using filter chamber arrays to obtain high-throughput DNA analysis.

  • 105.
    Russom, Aman
    et al.
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Tooke, Nigel
    Andersson, Helene
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Stemme, Göran
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Single nucleotide polymorphism analysis by allele-specific primer extension with real-time bioluminescence detection in a microfluidic device2003In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1014, no 1-2, p. 37-45Article in journal (Refereed)
    Abstract [en]

    A microfluidic approach for rapid bioluminescent real-time detection of single nucleotide polymorphism (SNP) is presented. The method is based on single-step primer extension using pyrosequencing chemistry to monitor nucleotide incorporations in real-time. The method takes advantage of the fact that the reaction kinetics differ between matched and mismatched primer-template configurations. We show here that monitoring the initial reaction in real time accurately scores SNPs by comparing the initial reaction kinetics between matched and mismatched configurations. Thus, no additional treatment is required to improve the sequence specificity of the extension, which has been the case for many allele-specific extension assays. The microfluidic approach was evaluated using four SNPs. Three of the SNPs included primer-template configurations that have been previously reported to be difficult to resolve by allele-specific primer extension. All SNPs investigated were successfully scored. Using the microfluidic device, the volume for the bioluminescent assay was reduced dramatically, thus offering a cost-effective and fast SNP analysis method.

  • 106.
    Rydholm, Susanna
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Frisk, Thomas
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Andersson, Helene
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Stemme, Göran
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Microfluidic device for studies of primary cilium direction sensitivity2005In: Proceedings of µTAS 2005 Conference, 2005, p. 1416-1418Conference paper (Refereed)
    Abstract [en]

    This paper presents a novel method for studying cilia forming cells in asymmetric microfluidic environments. It has previously been shown that bending the primary cilium by a fluid flow will give rise to a calcium signal, but the sensitivity for flow direction has so far not been studied. The microfluidic device presented here was designed for control of the local direction of fluid flow on the cellular level, and thus, enables studies of cellular response to a direction controlled cilium movement. Cells seeded on cover slips form cilia with the average length 2.9 μm after three days in culture and 4.3 μm after four days. Distinct calcium peaks were found after the initiation of flow in the channel. By using a microstructured flow system we have been able to study the sensitivity of confluent COS 7 cells expressing primary cilium to changes in fluid flow.

  • 107.
    Rydholm, Susanna
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Frisk, Thomas
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Andersson, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Stemme, Göran
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Three Dimensional Asymmetric Microenvironment for Cell Biological Studies2006Conference paper (Refereed)
  • 108.
    Rydholm, Susanna
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Frisk, Thomas
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Stemme, Göran
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Three Microfluidic Device for Studies of Primary Cilium Direction Sensitivity2006Conference paper (Refereed)
  • 109.
    Rydholm, Susanna
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Frisk, Thomas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Kowalewski, Jacob
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Stemme, Göran
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Controlled stimuli of primary cilia in microfabricated device2008Conference paper (Refereed)
  • 110.
    Rydholm, Susanna
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Frisk, Thomas
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Kowalewski, Jacob M
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Stemme, Göran
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Microfluidic devices for studies of primary cilium mediated cellular response to dynamic flow conditions2008In: Biomedical microdevices (Print), ISSN 1387-2176, E-ISSN 1572-8781, Vol. 10, no 4, p. 555-560Article in journal (Refereed)
    Abstract [en]

    We present the first microfabricated microfluidic devices designed specifically for studies of primary cilium mediated cellular response to dynamic flow. The primary cilium functions as a mechano-sensor in renal tubular epithelium, sensing the extracellular fluid flow. Malfunction of cilia has been implicated in e.g. polycystic kidney disease and other pathological conditions. Bending of the primary cilium by fluid flow has been shown to give rise to an intracellular calcium signal, however little is known about the sensitivity to flow duration, magnitude and direction. This paper presents a novel method for studying cilia forming cells in asymmetric microfluidic environments. The microfluidic devices presented here were designed for a dynamic control of the local fluid flow on a cellular level, and thus, enables studies of cellular responses to an amplitude, frequency and direction controlled cilium movement.

  • 111. Sato, Kae
    et al.
    Sasaki, Naoki
    Svahn, Helene Andersson
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Sato, Kiichi
    Microfluidics for nano-pathophysiology2014In: Advanced Drug Delivery Reviews, ISSN 0169-409X, E-ISSN 1872-8294, Vol. 74, p. 115-121Article, review/survey (Refereed)
    Abstract [en]

    Nanotechnology-based drug delivery systems hold promise for innovative medical treatment of cancers. While drug materials are constantly under development, there are no practical cell-based models to assess whether these materials can reach the target tissue. Recently developed microfluidic systems have revolutionized cell-based experiments. In these systems, vascular endothelial cells and interstitium are set in microchannels that mimic microvessels. Drug permeability can be assayed in these blood vessel models under fluidic conditions that mimic blood flow. In this review, we describe device fabrication, disease model development, nanoparticle permeability assays, and the potential utility of these systems in the future.

  • 112.
    Sjöström, Staffan
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Huang, Mingtao
    Nielsen, Jens
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Jönsson, Håkan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Micro-droplet based directed evolution outperforms conventional laboratory evolution2014In: 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014, Chemical and Biological Microsystems Society , 2014, p. 169-171Conference paper (Refereed)
    Abstract [en]

    We present droplet adaptive laboratory evolution (DrALE), a directed evolution method used to improve industrial enzyme producing microorganisms for e.g. feedstock digestion. DrALE is based linking a desired phenotype to growth rate allowing only desired cells to proliferate. Single cells are confined in microfluidic droplets to prevent the phenotype, e.g. secreted enzymes, from leaking between cells. The method was benchmarked against and found to significantly outperform conventional adaptive laboratory evolution (ALE) in enriching enzyme producing cells. It was furthermore applied to enrich a whole-genome mutated library of yeast cells for α-amylase activity.

  • 113.
    Sjöström, Staffan L.
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Bai, Yunpeng
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Huang, Mingtao
    Liu, Zihe
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Jönsson, Håkan N.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    High-throughput screening for industrial enzyme production hosts by droplet microfluidics2014In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 14, no 4, p. 806-813Article in journal (Refereed)
    Abstract [en]

    A high-throughput method for single cell screening by microfluidic droplet sorting is applied to a whole-genome mutated yeast cell library yielding improved production hosts of secreted industrial enzymes. The sorting method is validated by enriching a yeast strain 14 times based on its a-amylase production, close to the theoretical maximum enrichment. Furthermore, a 105 member yeast cell library is screened yielding a clone with a more than 2-fold increase in a-amylase production. The increase in enzyme production results from an improvement of the cellular functions of the production host in contrast to previous droplet-based directed evolution that has focused on improving enzyme protein structure. In the workflow presented, enzyme producing single cells are encapsulated in 20 pL droplets with a fluorogenic reporter substrate. The coupling of a desired phenotype (secreted enzyme concentration) with the genotype (contained in the cell) inside a droplet enables selection of single cells with improved enzyme production capacity by droplet sorting. The platform has a throughput over 300 times higher than that of the current industry standard, an automated microtiter plate screening system. At the same time, reagent consumption for a screening experiment is decreased a million fold, greatly reducing the costs of evolutionary engineering of production strains.

  • 114.
    Sjöström, Staffan L.
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Jönsson, Håkan N.
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Multiplex analysis of enzyme kinetics and inhibition by droplet microfluidics using picoinjectors2013In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 13, no 9, p. 1754-1761Article in journal (Refereed)
    Abstract [en]

    Enzyme kinetics and inhibition is important for a wide range of disciplines including pharmacology, medicine and industrial bioprocess technology. We present a novel microdroplet-based device for extensive characterization of the reaction kinetics of enzyme substrate inhibitor systems in a single experiment utilizing an integrated droplet picoinjector for bioanalysis. This device enables the scanning of multiple fluorescently-barcoded inhibitor concentrations and substrate conditions in a single, highly time-resolved experiment yielding the Michaelis constant (K-m), the turnover number (k(cat)) and the enzyme inhibitor dissociation constants (k(i), k(i)'). Using this device we determine K-m and k(cat) for beta-galactosidase and the fluorogenic substrate Resorufin beta-D-galactopyranoside (RBG) to be 442 mu M and 1070 s(-1), respectively. Furthermore, we examine the inhibitory effects of isopropyl-beta-D-thiogalactopyranoside (IPTG) on beta-galactosidase. This system has a number of potential applications, for example it could be used to screen inhibitors to pharmaceutically relevant enzymes and to characterize engineered enzyme variants for biofuels production, in both cases acquiring detailed information about the enzyme catalysis and enzyme inhibitor interaction at high throughput and low cost.

  • 115.
    Sjöström, Staffan L.
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Jönsson, Håkan N.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    High-­throughput screening for improved enzymes in environments lethal to host cellsManuscript (preprint) (Other academic)
  • 116.
    Sjöström, Staffan L.
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Jönsson, Håkan N.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Svahn, Helene Andersson
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Multiplex analysis of enzyme kinetics and inhibition by droplet microfluidics using picoinjectors2012In: Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, 2012, p. 172-174Conference paper (Refereed)
    Abstract [en]

    We present a novel microdroplet-based device for extensive characterization of the reaction kinetics of enzymeinhibitor systems in a single experiment, for the first time utilizing droplet picoinjectors for bioanalysis. This device enables the scanning of multiple inhibitors, inhibitor concentrations and substrate conditions in a single, highly time resolved experiment yielding the Michaelis constant (Km), the turnover number (Kcat) the mode of inhibition and the inhibitor enzyme binding constants (Ki, Ki). Using this device we determine Km and Kcat for β-galactosidase and the fluorogenic substrate Resorufin β-D-galactopyranoside (RBG) to 252 μM and 477 s-1, respectively. Furthermore, we examine the inhibitory effects of Phenylethyl β-D-thiogalactopyranoside (PETG) on this system.

  • 117.
    Stemme, Göran
    et al.
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Frisk, Thomas
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Rydholm, Susanna
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Andersson, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    A cell-relevant Microgradient Environment2006In: Proceedings of the 10th International Conference in Miniaturized Systems for Chemistry and Life Sciences (µTas), 2006, p. 1507-1509Conference paper (Refereed)
    Abstract [en]

    With confocal microscopy new knowledge in cell physiology is acquireddaily. However, most cell assays today are carried out either as multiwellplate assays, or in standard petridish assays. These two methods havedifferent features and foci, but they have in common the large amount ofcells submitted for treatment and imaging. In order to study only a few cellson a more detailed level[1, 2] in a relevant context, we have designed, built,and evaluated a microfluidic system. It features 1) immobilization of cells inthree dimensions, 2) transportation of cell nutrients and treatments as wellas removal of residual products, 3) an extremely stable and physiologicallyrelevant gradient of chemical concentration distribution around the cell.Previous efforts in this field by our group revealed a few very importantissues, indicating that microfabrication would be the enabling technologyfor experiments on cells in asymmetricenvironments.

  • 118.
    Stemme, Göran
    et al.
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Rydholm, Susanna
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Frisk, Thomas
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Andersson, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Microfabricated Device for Controlled Stimuli of Primary Cilia2006In: Proceedings of the 10th International Conference in Miniaturized Systems for Chemistry and Life Sciences (µTas), 2006, p. 1510-1512Conference paper (Refereed)
    Abstract [en]

    We present an improved device and method for studying cellular response onmicrofluidic controlled stimuli of primary cilia. The primary cilium functions as amechano-sensor in renal tubular epithelium. Malfunction of cilia has been implicated inpolycystic kidney decease as well as other kidney abnormalities. Bending of cilia willgive rise to an intracellular calcium signal [1,2,3], but little is known about theimportance of flow direction, magnitude and duration to the calcium response. Ourpreliminary results indicate flow speed sensitivity.

  • 119.
    Ståhl, Patrik L.
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Gantelius, Jesper
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Natanaelsson, Christian
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Visual DNA: Identification of DNA sequence variations by bead trapping2007In: Genomics, ISSN 0888-7543, E-ISSN 1089-8646, Vol. 90, p. 741-745Article in journal (Refereed)
    Abstract [en]

    In this paper we describe a method that uses the nearly covalent strength biotin-streptavidin interaction to attach a paramagnetic bead of micrometer size to a DNA molecule of nanometer size, scaling up the spatial size of a query DNA strand by a factor of 1000, making it visible to the human eye. The use of magnetic principles enables rapid binding and washing of detector beads, facilitating a readout of amplified DNA sequences in a few minutes. Here we exemplify the method on mitochondrial DNA variations using an array platform. Visual identification and documentation can be performed with ail ordinary mobile phone equipped with a built-in camera.

  • 120.
    Svedberg, Gustav
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. Science for Life Laboratory.
    Gantelius, Jesper
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. Science for Life Laboratory.
    Svahn, Helene Andersson
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. Science for Life Laboratory.
    A printer-free, vertical flow based, colorimetric planar bead array for point of care applications2015In: MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Chemical and Biological Microsystems Society , 2015, p. 945-947Conference paper (Refereed)
    Abstract [en]

    We present a novel planar bead array that utilizes fluorescent colour coding, a convenient and quick bead immobilization technique, rapid pump-driven sample delivery and colorimetric readout enabling analysis of the array using an inexpensive USB microscope or smartphone camera. The array combines high multiplexing potential with a low assay run time and point of care amenability due to low equipment requirements.

  • 121.
    Svedberg, Gustav
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Jeong, Yunjin
    Na, Hunjong
    Jang, Jisung
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kwon, Sunghoon
    Gantelius, Jesper
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Svahn Andersson, Helene
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Towards encoded particles for highly multiplexed colorimetric point of care autoantibody detection2017In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 17, no 3, p. 549-556Article in journal (Refereed)
    Abstract [en]

    Highly multiplexed point of care tests could improve diagnostic accuracy and differential diagnostic capacity in for instance emergency medicine and low resource environments. Available technology platforms for POC biomarker detection are typically simplex or low-plexed, whereas common lab-based microarray systems allow for the simultaneous detection of thousands of DNA or protein biomarkers. In this study, we demonstrate a novel suspension particle array platform that utilizes 900 mu m bricks for POC amenable colorimetric biomarker detection with an encoding capacity of over two million. Due to the mm-scale size, both the lithographic codes and colorimetric signals of individual particles can be visualized using a consumer grade office flatbed scanner, with a potential for simultaneous imaging of around 19000 particles per scan. The analytical sensitivity of the assay was determined to be 4 ng ml(-1) using an antibody model system. As a proof of concept, autoantibodies toward anoctamin 2 were detected in order to discriminate between multiple sclerosis plasma samples and healthy controls with p < 0.0001 and an inter-assay % CV of 9.44%.

  • 122.
    Söderberg, Lovisa
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Jönsson, Håkan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Parallel cDNA synthesis from thousands of individually encapsulated cancer cells: Towards large scale single cell gene expression analysis2013In: 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013, 2013, Vol. 3, p. 1737-1739Conference paper (Refereed)
    Abstract [en]

    We present microfluidic droplet-based cDNA synthesis of 65 000 individually isolated lung cancer cells in parallel. Cells are encapsulated, individually lysed and the RNA from each cell is reverse transcribed in droplets at a massively parallel scale resulting in thousands of droplets each containing the cells gene expression profile encoded in stable DNA for downstream analysis (figure 1). This could be used for distinguishing between different cell types and study heterogeneity within a cell sample at high throughput scale.

  • 123.
    Uhlén, Mathias
    et al.
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Affinity reagents for lab on chips2011In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 11, no 8, p. 1417-1419Article in journal (Other academic)
  • 124.
    Uhlén, Mathias
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Svahn, Helene Andersson
    KTH, School of Biotechnology (BIO), Proteomics.
    Lab on a chip technologies for bioenergy and biosustainability research2011In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 11, no 20, p. 3389-3393Article in journal (Other academic)
  • 125. Valero, A.
    et al.
    Merino, F.
    Wolbers, F.
    Luttge, R.
    Vermes, I.
    Andersson, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    van den Berg, A.
    Apoptotic cell death dynamics of HL60 cells studied using a microfluidic cell trap device2005In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 5, no 1, p. 49-55Article in journal (Refereed)
    Abstract [en]

    This paper presents the design, fabrication and first results of a microfluidic cell trap device for analysis of apoptosis. The microfluidic silicon-glass chip enables the immobilization of cells and real-time monitoring of the apoptotic process. Induction of apoptosis, either electric field mediated or chemically induced with tumour necrosis factor (TNF-alpha), in combination with cycloheximide (CHX), was addressed. Exposure of cells to the appropriate fluorescent dyes, FLICA and PI, allows one to discriminate between viable, apoptotic and necrotic cells. The results showed that the onset of apoptosis and the transitions during the course of the cell death cascade were followed in chemically induced apoptotic HL60 cells. For the case of electric field mediated cell death, the distinction between apoptotic and necrotic stage was not clear. This paper presents the first results to analyse programmed cell death dynamics using this apoptosis chip and a first step towards an integrated apoptosis chip for high-throughput drug screening on a single cellular level.

  • 126.
    Vanherberghen, Bruno
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Kowalewski, Jacob
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Garrod, Kym
    Lindström, Sara
    KTH, School of Biotechnology (BIO).
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO).
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Cahalan, Michael D.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Single Cell Tracking of Natural Killer CellMigration in vivo and in vitro reveals Transient Migration Arrest PeriodsManuscript (preprint) (Other (popular science, discussion, etc.))
  • 127.
    Veses-Garcia, Marta
    et al.
    Karolinska Inst, Swedish Med Nanosci Ctr, Dept Neurosci, Stockholm, Sweden..
    Antypas, Haris
    Karolinska Inst, Swedish Med Nanosci Ctr, Dept Neurosci, Stockholm, Sweden..
    Loffler, Susanne
    Karolinska Inst, Swedish Med Nanosci Ctr, Dept Neurosci, Stockholm, Sweden..
    Brauner, Annelie
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Div Clin Microbiol, Stockholm, Sweden.;Karolinska Univ Hosp, Stockholm, Sweden..
    Svahn Andersson, Helene
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Richter-Dahlfors, Agneta
    Karolinska Inst, Swedish Med Nanosci Ctr, Dept Neurosci, Stockholm, Sweden..
    Rapid Phenotypic Antibiotic Susceptibility Testing of Uropathogens Using Optical Signal Analysis on the Nanowell Slide2018In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 9, article id 1530Article in journal (Refereed)
    Abstract [en]

    Achieving fast antimicrobial susceptibility results is a primary goal in the fight against antimicrobial resistance. Standard antibiotic susceptibility testing (AST) takes, however, at least a day from patient sample to susceptibility profile. Here, we developed and clinically validated a rapid phenotypic AST based on a miniaturized nanotiter plate, the nanowell slide, that holds 672 wells in a 500 nl format for bacterial cultivation. The multitude of nanowells allows multiplexing with a panel of six antibiotics relevant for urinary tract infections. Inclusion of seven concentrations per antibiotic plus technical replicates enabled us to determine a precise minimum inhibitory concentration for 70 clinical uropathogenic Escherichia coil isolates. By combining optical recordings of bacterial growth with an algorithm for optical signal analysis, we calculated T-lag, the point of transition from lag to exponential phase, in each nanoculture. Algorithm-assisted analysis determined antibiotic susceptibility as early as 3 h 40 min. In comparison to standard disk diffusion assays, the nanowell AST showed a total categorical agreement of 97.9% with 2.6% major errors and 0% very major errors for all isolate-antibiotic combination tested. Taking advantage of the optical compatibility of the nanowell slide, we performed microscopy to illustrate its potential in defining susceptibility profiles based on bacterial morphotyping. The excellent clinical performance of the nanowell AST, combined with a short detection time, morphotyping, and the very low consumption of reagents clearly show the advantage of this phenotypic AST as a diagnostic tool in a clinical setting.

  • 128.
    Weibull, Emilie
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ohashi, Toshiro
    A microfluidic device towards shear stress analysis on clonal expanded endothelial cells2013Manuscript (preprint) (Other academic)
  • 129.
    Weibull, Emilie
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Antipas, Haris
    Richter-Dahlfors, Agneta
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Dynamic optical screening of single-bacterium and retrieval of the subsequent liquid colony for genetic analysis2014Manuscript (preprint) (Other academic)
  • 130.
    Weibull, Emilie
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Antypas, Haris
    Kjall, Peter
    Brauner, Annelie
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Richter-Dahlfors, Agneta
    Bacterial Nanoscale Cultures for Phenotypic Multiplexed Antibiotic Susceptibility Testing2014In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 52, no 9, p. 3310-3317Article in journal (Refereed)
    Abstract [en]

    An optimal antimicrobial drug regimen is the key to successful clinical outcomes of bacterial infections. To direct the choice of antibiotic, access to fast and precise antibiotic susceptibility profiling of the infecting bacteria is critical. We have developed a high-throughput nanowell antibiotic susceptibility testing (AST) device for direct, multiplexed analysis. By processing in real time the optical recordings of nanoscale cultures of reference and clinical uropathogenic Escherichia coli strains with a mathematical algorithm, the time point when growth shifts from lag phase to early logarithmic phase (T-lag) was identified for each of the several hundreds of cultures tested. Based on T-lag, the MIC could be defined within 4 h. Heatmap presentation of data from this high-throughput analysis allowed multiple resistance patterns to be differentiated at a glance. With a possibility to enhance multiplexing capacity, this device serves as a high-throughput diagnostic tool that rapidly aids clinicians in prescribing the optimal antibiotic therapy.

  • 131.
    Weibull, Emilie
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Antypas, Haris
    Kjäll, Peter
    Brauner, Annelie
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Richter-Dahlfors, Agneta
    Bacterial nano-scale cultures for rapid multiplexed antibiotic susceptibility testing2013Manuscript (preprint) (Other academic)
  • 132.
    Weibull, Emilie
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Bai, Yunpeng
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Jönsson, Håkan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Andersson Svahn, Helen
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Interfacing picoliter droplet microfluidics with addressable μl-compartments using FACS2013In: 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013, 2013, Vol. 3, p. 1632-1634Conference paper (Refereed)
    Abstract [en]

    We present a high-throughput technique to interface picoliter droplet microfluidics for single cell analysis with a macro scale accessible array platform by the addition of an agarose gelling agent to droplets and patterned positioning of the resulting hydrogel beads using a fluorescence activated cell sorter (FACS). This resulted in a pattern with 95 % single bead accuracy. Agarose beads containing eGFP expressing E. Coli were single sorted into microwells and E. coli growth was monitored over time.

  • 133.
    Weibull, Emilie
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Lindström, S.
    Segerman, A.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Towards automated high content screening on a 672-microwell slide2010In: 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010: Volume 2, 2010, p. 965-967Conference paper (Refereed)
    Abstract [en]

    We present an automated high content screening method for single-cell analysis on a microwell slide. The 672 wells are screened in 73 minutes, i.e. a 10-fold increase, yielding a negative/positive response using an automated confocal software. This screening method is highly sophisticated, rapid and results in high-resolution imaging. The next step is to apply the method to a heterogeneous glioma cell line using a stem cell- and astrocyte marker to study the link between marker expression and proliferation rate on single cells. The resulting images are input files in a cell analyzing software to attain information about cell density, co-localization, cell size and cell count.

  • 134.
    Weibull, Emilie
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Matsui, S.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Ohashi, T.
    Development of a microfluidic concentration gradient generator on a microwell slide for high-throughput cell analysis2012In: Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, Chemical and Biological Microsystems Society , 2012, p. 1573-1575Conference paper (Refereed)
    Abstract [en]

    The present study reports a newly developed, microfluidic concentration gradient generator that adds substantial utility to the high-throughput nature of the microwell slide that we have previously established [1, 2]. Using microfluidic dynamics, we have designed microchannels to deliver a reagent and its dilutant with precisely controlled flow volumes, generating 8 discrete steps of reagent concentration in designated microwells. As proof of concept FITC-labeled dextran and dextran-free water was used to visualize the concentration gradient. The next step will be to perform cancer drug concentration gradient experiments on an animal cell line.

  • 135.
    Weibull, Emilie
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Matsui, S.
    Sakai, M.
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ohashi, T.
    Microfluidic device for generating a stepwise concentration gradient on a microwell slide for cell analysis2013In: Biomicrofluidics, ISSN 1932-1058, E-ISSN 1932-1058, Vol. 7, no 6, p. 064115-Article in journal (Refereed)
    Abstract [en]

    Understanding biomolecular gradients and their role in biological processes is essential for fully comprehending the underlying mechanisms of cells in living tissue. Conventional in vitro gradient-generating methods are unpredictable and difficult to characterize, owing to temporal and spatial fluctuations. The field of microfluidics enables complex user-defined gradients to be generated based on a detailed understanding of fluidic behavior at the μm-scale. By using microfluidic gradients created by flow, it is possible to develop rapid and dynamic stepwise concentration gradients. However, cells exposed to stepwise gradients can be perturbed by signals from neighboring cells exposed to another concentration. Hence, there is a need for a device that generates a stepwise gradient at discrete and isolated locations. Here, we present a microfluidic device for generating a stepwise concentration gradient, which utilizes a microwell slide’s pre-defined compartmentalized structure to physically separate different reagent concentrations. The gradient was generated due to flow resistance in the microchannel configuration of the device, which was designed using hydraulic analogy and theoretically verified by computational fluidic dynamics simulations. The device had two reagent channels and two dilutant channels, leading to eight chambers, each containing 4 microwells. A dose-dependency assay was performed using bovine aortic endothelial cells treated with saponin. High reproducibility between experiments was confirmed by evaluating the number of living cells in a live-dead assay. Our device generates a fully mixed fluid profile using a simple microchannel configuration and could be used in various gradient studies, e.g., screening for cytostatics or antibiotics.

  • 136. Wolbers, F.
    et al.
    Andersson, Helene
    KTH, Superseded Departments, Biotechnology.
    van den Berg, A.
    Vermes, I.
    Apoptosis induced kinetic changes in autofluorescence of cultured HL60 cells-possible application for single cell analysis on chip2004In: Apoptosis (London), ISSN 1360-8185, E-ISSN 1573-675X, Vol. 9, no 6, p. 749-755Article in journal (Refereed)
    Abstract [en]

    Introduction: This paper presents a new method using natural cellular fluorescence (autofluorescence, AF) to study apoptosis. Measurement of AF reduces sample preparation time and avoids cellular toxicity due to the fact that no labelling is required. Methods: Human promyelocytic leukemic HL60 cells were incubated with camptothecin (CPT), tumour necrosis factor (TNF)-alpha in combination with cycloheximide (CHX), or irradiated with 6 or 10 Gray, during varying time periods, to initiate apoptosis. AF was measured at the flow cytometer. Results: Induction of apoptosis results in the shrinkage of the cell and the fragmentation into apoptotic bodies. With flow cytometry, 4 subpopulations, viable, early apoptotic, late apoptotic and the necrotic cells, can be distinguished. Induction of apoptosis results in a decrease in AF intensity compared to untreated HL60 cells, especially seen in the late apoptotic subpopulation. The AF intensity is found to decrease significantly in time (between 2 h and 24 h) for all the four apoptotic inducers used. Conclusions: Our results show that it is possible to specifically measure the apoptotic-induced kinetic changes in AF in HL60 cells. A decrease in AF intensity is seen from 2 h till 24 h. These results open a door for future developments in single-cell analysis.

  • 137. Wolbers, F.
    et al.
    Franke, H. R.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Vermes, I.
    Van Den Berg, A.
    Microfluidic apoptosis chip for drug screening to improve and personalize cancer therapy2008In: 12th International Conference on Miniaturized Systems for Chemistry and Life Sciences - The Proceedings of MicroTAS 2008 Conference, Chemical and Biological Microsystems Society , 2008, p. 549-551Conference paper (Refereed)
    Abstract [en]

    Currently, the knowledge on breast cancer treatment is increasing, however, there are still hardly any assays present to match patients to the right form of therapy to enhance the therapeutic effectiveness. Therefore a microfluidic device is developed to analyze the hormonal and chemosensitivity of first breast cancer cell lines (MCF-7) and in a later stage a patients' own tumor cells. The apoptotic inducers tumor necrosis factor-α, in combination with cycloheximide, and staurosporine stained MCF-7 cells positive for Annexin V and PI, and showed a decrease in area coverage as compared to untreated cells.

123 101 - 137 of 137
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