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  • 1.
    Ahmadian, Afshin
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    AnderssonSvahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Massively parallel sequencing platforms using lab on a chip technologies2011In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 11, no 16, p. 2653-2655Article in journal (Refereed)
  • 2.
    Andersson, Helene
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    van den Berg, Albert
    KTH.
    Where are the biologists?: A series of mini-reviews covering new trends in fundamental and applied research, and potential applications of miniaturised technologies2006In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 6, no 4, p. 467-470Article in journal (Refereed)
  • 3.
    Ardabili, Sahar
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Gantelius, Jesper
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Kowalewski, Jacob
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Russom, Aman
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Dean flow-coupled inertial focusing for ultra-high-throughput particle filtration2010In: 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010: Volume 3, 2010, p. 1586-1588Conference paper (Refereed)
    Abstract [en]

    Particle manipulation represents an important and fundamental step prior to counting, sorting and detecting bio-particles. In this study, we report dean-coupled inertial focusing of particles in flows through a single curve microchannel at extremely high channel Reynold numbers (∼325). We found the lateral particle focusing position, xf to be fixed and largely independent of radius of curvature and whether particles are pre-focused (at equilibrium) entering the curvature or randomly distributed. Finally, using a single inlet, u-shaped, microchannel we demonstrate filtration of 10μm particles from 2 μm particles at throughputs several orders of magnitude higher than previously shown.

  • 4. DaCosta, R. S.
    et al.
    Andersson, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Cirocco, M.
    Marcon, N. E.
    Wilson, B. C.
    Autofluorescence characterisation of isolated whole crypts and primary cultured human epithelial cells from normal, hyperplastic, and adenomatous colonic mucosa2005In: Journal of Clinical Pathology, ISSN 0021-9746, E-ISSN 1472-4146, Vol. 58, no 7, p. 766-774Article in journal (Refereed)
    Abstract [en]

    Background/Aims: In vivo autofluorescence endoscopic imaging and spectroscopy have been used to detect and differentiate benign ( hyperplastic) and preneoplastic ( adenomatous) colonic lesions. This fluorescence is composed of contributions from the epithelium, lamina propria, and submucosa. Because epithelial autofluorescence in normal and diseased tissues is poorly understood, this was the focus of the present study. Methods: Whole colonic crypts were isolated, and short term primary cultures of epithelial cells were established from biopsies of normal, hyperplastic, and adenomatous colon. Autofluorescence ( 488 nm excitation) was examined by confocal fluorescence microscopy. Fluorescently labelled organelle probes and transmission electron microscopy were used to identify subcellular sources of fluorescence. Results: Mitochondria and lysosomes were identified as the main intracellular fluorescent components in all cell types. Normal and hyperplastic epithelial cells were weakly autofluorescent and had similar numbers of mitochondria and lysosomes, whereas adenomatous ( dysplastic) epithelial cells showed much higher autofluorescence, and numerous highly autofluorescent lysosomal ( lipofuscin) granules. Conclusions: Short term primary cell cultures from endoscopic biopsies provide a novel model to understand differences in colonic tissue autofluorescence at the glandular ( crypt) and cellular levels. The differences between normal, hyperplastic, and adenomatous epithelial cells are attributed in part to differences in the intrinsic numbers of mitochondria and lysosomes. This suggests that the detection of colonic epithelial fluorescence alone, if possible, may be sufficient to differentiate benign ( hyperplastic) from preneoplastic and neoplastic ( adenomatous) colonic intramucosal lesions during in vivo fluorescence endoscopy. Furthermore, highly orange/red autofluorescent intracellular granules found only in dysplastic epithelial cells may serve as a potential biomarker.

  • 5.
    Friedman, Mikaela
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Lindström, Sara
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Engineering and characterization of a bispecific HER2 × EGFR-binding affibody molecule2009In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 54, p. 121-131Article in journal (Refereed)
    Abstract [en]

    HER2 (human epidermal-growth-factor receptor-2; ErbB2) and EGFR (epidermal-growth-factor receptor) are overexpressed in various forms of cancer, and the co-expression of both HER2 and EGFR has been reported in a number of studies. The simultaneous targeting of HER2 and EGFR has been discussed as a strategy with which to potentially increase efficiency and selectivity in molecular imaging and therapy of certain cancers. In an effort to generate a molecule capable of bispecifically targeting HER2 and EGFR, a gene fragment encoding a bivalent HER2-binding affibody molecule was genetically fused in-frame with a bivalent EGFR-binding affibody molecule via a (G(4)S)(3) [(Gly(4)-Ser)(3)]-encoding gene fragment. The encoded 30 kDa affibody construct (Z(HER2))(2)-(G(4)S)(3)-(Z(EGFR))(2), with potential for bs (bispecific) binding to HER2 and EGFR, was expressed in Escherichia coli and characterized in terms of its binding capabilities. The retained ability to bind HER2 and EGFR separately was demonstrated using both biosensor technology and flow-cytometric analysis, the latter using HER2- and EGFR-overexpressing cells. Furthermore, simultaneous binding to HER2 and EGFR was demonstrated in: (i) a sandwich format employing real-time biospecific interaction analysis where the bs affibody molecule bound immobilized EGFR and soluble HER2; (ii) immunofluorescence microscopy, where the bs affibody molecule bound EGFR-overexpressing cells and soluble HER2; and (iii) a cell-cell interaction analysis where the bs affibody molecule bound HER2-overexpressing SKBR-3 cells and EGFR-overexpressing A-431 cells. This is, to our knowledge, the first reported bs affinity protein with potential ability for the simultaneous targeting of HER2 and EGFR. The potential future use of this and similar constructs, capable of bs targeting of receptors to increase the efficacy and selectivity in imaging and therapy, is discussed.

  • 6.
    Frisk, Thomas
    et al.
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Rydholm, Susanna
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Stemme, Göran
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Three dimensional asymmetric microenvironment for cell biologigal studies2005In: Proceedings Micro Total Analysis Systems (muTAS) 2005, 2005Conference paper (Refereed)
  • 7.
    Frisk, Thomas
    et al.
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Rydholm, Susanna
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Liebmann, Thomas
    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.
    A microfluidic device for parallel 3-D cell cultures in asymmetric environments2007In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 28, no 24, p. 4705-4712Article in journal (Refereed)
    Abstract [en]

    We demonstrate a concept for how a miniaturized 3-D cell culture in biological extracellular matrix (ECM) or synthetic gels bridges the gap between organ-tissue culture and traditional 2-D cultures. A microfluidic device for 3-D cell culture including microgradient environments has been designed, fabricated, and successfully evaluated. In the presented system stable diffusion gradients can be generated by application of two parallel fluid flows with different composition against opposite sides of a gel plug with embedded cello. Culture for up to two weeks was performed showing cells still viable and proliferating. The cell tracer dye calcein was used to verify gradient formation as the fluorescence intensity in exposed cells was proportional to the position in the chamber. Cellular response to an applied stimulus was demonstrated by use of an adenosine triphosphate gradient where the onset of a stimulated intracellular calcium release also depended on cell position.

  • 8.
    Gantelius, Jesper
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Novel diagnostic microarray assay formats towards comprehensive on-site analysis2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Advances in molecular methods for analyzing DNA, RNA and proteins in humans as well as in other animals, plants, fungi, bacteria or viruses have greatly increased the resolution with which we can study life’s complexity and dynamics on earth. While genomic, transcriptomic and proteomic laboratory tools for molecular diagnosis of disease are rapidly becoming more comprehensive, the access to such advanced yet often expensive and centralized procedures is limited. There is a great need for rapid and comprehensive diagnostic methods in low-resource settings or contexts where a person can not or will not go to a hospital or medical laboratory, yet where a clinical analysis is urgent.

    In this thesis, results from development and characterization of novel technologies for DNA and protein microarray analysis are presented. Emphasis is on methods that could provide rapid, cost-effective and portable analysis with convenient readout and retained diagnostic accuracy. The first study presents a magnetic bead-based approach for DNA microarray analysis for a rapid visual detection of single nucleotide polymorphisms. In the second work, magnetic beads were used as detection reagents for rapid differential detection of presence of pestiviral family members using a DNA oligonucleotide microarray with read-out by means of a tabletop scanner or a digital camera. In paper three, autoimmune responses from human sera were detected on a protein autoantigen microarray, again by means of magnetic bead analysis. Here, special emphasis was made in comprehensively comparing the performance of the magnetic bead detection to common fluorescence-based detection. In the fourth study, an immunochromatographic lateral flow protein microarray assay is presented for application in the classification of contagious pleuropneumonia from bovine serum samples. The analysis could be performed within 10 minutes using a table top scanner, and the performance of the assay was shown to be comparable to that of a cocktail ELISA. In the fifth paper, the lateral flow microarray framework is investigated in further detail by means of experiments and numerical simulation. It was found that downstream effects play an important role, and the results further suggest that the downstream binding profiles may find use in simple affinity evaluation.

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  • 9.
    Gantelius, Jesper
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Bass, Tarek
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Gundberg, Anna
    KTH, School of Biotechnology (BIO), Proteomics.
    Sundberg, Mårten
    KTH, School of Biotechnology (BIO), Proteomics.
    Sjöberg, Ronald
    KTH, School of Biotechnology (BIO), Proteomics.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    A ten-minute high density lateral flow protein microarray assay2011In: 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011: (MicroTAS 2011), 2011, p. 1176-1178Conference paper (Refereed)
    Abstract [en]

    Protein microarrays are useful tools for highly multiplexed determination of presence or levels of clinically relevant biomarkers in human tissues and biofluids. However, such tools have thus far been restricted to laboratory environments. Here, we present a novel 384-plexed easy to use lateral flow protein microarray device capable of sensitive (<50ng/ml) determination of antigen specific antibodies in less than ten minutes total assay time. Results were developed with gold nanobeads and could be recorded by a cell-phone camera or table top scanner. Excellent accuracy (AUC=99.4%) was achieved in comparison with an established glass microarray assay for 26 antigen-specific antibodies.

  • 10.
    Gantelius, Jesper
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Bass, Tarek
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Sjöberg, Ronald
    KTH, School of Biotechnology (BIO), Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    A Lateral Flow Protein Microarray for Rapid and Sensitive Antibody Assays2011In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 12, no 11, p. 7748-7759Article in journal (Refereed)
    Abstract [en]

    Protein microarrays are useful tools for highly multiplexed determination of presence or levels of clinically relevant biomarkers in human tissues and biofluids. However, such tools have thus far been restricted to laboratory environments. Here, we present a novel 384-plexed easy to use lateral flow protein microarray device capable of sensitive (<30 ng/mL) determination of antigen-specific antibodies in ten minutes of total assay time. Results were developed with gold nanobeads and could be recorded by a cell-phone camera or table top scanner. Excellent accuracy with an area under curve (AUC of 98% was achieved in comparison with an established glass microarray assay for 26 antigen-specific antibodies. We propose that the presented framework could find use in convenient and cost-efficient quality control of antibody production, as well as in providing a platform for multiplexed affinity-based assays in low-resource or mobile settings.

  • 11.
    Gantelius, Jesper
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Hartmann, Michael
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics.
    Roeraade, Johan
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Joos, Thomas O
    Magnetic bead-based detection of autoimmune responses using protein microarrays.2009In: New biotechnology, ISSN 1871-6784, Vol. 26, p. 269-276Article in journal (Refereed)
    Abstract [en]

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

  • 12.
    Gantelius, Jesper
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Härlin, A.
    Elfversson, G.
    Nystrand, M.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Improved sensitivity on an allergen lateral flow microarray by means of dendritic amplification2009In: Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Chemical and Biological Microsystems Society , 2009, p. 1136-1137Conference paper (Refereed)
    Abstract [en]

    Recently, paper-based substrates have been proposed as an alternative to commonly used activated glass slides for microarray patterning[1], used in conjunction with capillary driven lateral flow of sample and detection reagents through the membrane. While fluorescent detection reagents may be employed to achieve high sensitivity, gold nanoparticles can also be used to allow readout by means of common table top scanners or digital cameras. Here, we demonstrate first results from employing a dendritic, or layer-by-layer, amplification approach for a high-density lateral flow allergen protein microarray, indicating that substantially increased sensitivity can be achieved with very modest increase of assay handling and time requirements.

  • 13.
    Gantelius, Jesper
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Nystrand, M.
    Harlin, A.
    Elfverson, G.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics.
    Uhlén, Mattias
    KTH, School of Biotechnology (BIO), Proteomics.
    Eriksson-Karlström, Amelie
    KTH, School of Biotechnology (BIO).
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Evaluation of a lateral flow microarray assay systemArticle in journal (Other academic)
  • 14.
    Gantelius, Jesper
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics.
    Hamsten, Carl
    KTH, School of Biotechnology (BIO).
    Neiman, Maja
    KTH, School of Biotechnology (BIO), Proteomics.
    Persson, Anja
    KTH, School of Biotechnology (BIO), Proteomics.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO).
    A lateral flow protein microarray for rapid determination of contagious bovine pleuropneumonia status in bovine serum2010In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 82, no 1, p. 11-18Article in journal (Refereed)
    Abstract [en]

    Novel analytical methods for a next generation of diagnostic devices combine attributes from sensitive, accurate, fast, simple and multiplexed analysis methods. Here, we describe a possible contribution to these by the application of a lateral flow microarray where a panel of recombinant protein antigens was used to differentiate bovine serum samples in the context of the lung disease contagious bovine pleuropneumonia (CBPP). Lateral flow arrays were produced by attaching nitrocellulose onto microscopic slides and spotting of the recombinant proteins onto the membranes. The developed assay included evaluations of substrate matrix and detection reagents to allow for short assay times and convenient read-out options, and to yield a total assay time from sample application to data acquisition of less than ten minutes. It was found that healthy and disease-affected animals could be discriminated (AUC = 97%), and we suggest that the use of an antigen panel in combination with the lateral flow device offers an emerging analytical tool towards a simplified but accurate on-site diagnosis.

  • 15.
    Guldevall, Karolin
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Vanherberghen, Bruno
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Frisk, Thomas
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet.
    Hurtig, Johan
    Department of Chemsitry, University of Washington, Seattle, USA.
    Christakou, Athanasia
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Manneberg, Otto
    Department of Environmental Health, Harvard School of Public Health, Boston, USA.
    Lindström, Sara
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Imaging Immune Surveillance of Individual Natural Killer Cells Confined in Microwell Arrays2010In: PLOS ONE, ISSN 1932-6203, Vol. 5, no 11, p. e15453-Article in journal (Refereed)
    Abstract [en]

    New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level.

  • 16.
    Hansson, Jonas
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Karlsson, Mikael J.
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Haraldsson, Tommy
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    van der Wijngaart, Wouter
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Russom, Aman
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Inertial microfluidics in parallel channels for high-throughput applications2012In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 12, no 22, p. 4644-4650Article in journal (Refereed)
    Abstract [en]

    Passive particle focusing based on inertial microfluidics was recently introduced as a high-throughput alternative to active focusing methods that require an external force-field to manipulate particles. In this study, we introduce inertial microfluidics in flows through straight, multiple parallel channels. The scalable, single inlet and two outlet, parallel channel system is enabled by a novel, high-density 3D PDMS microchannel manufacturing technology, mediated via a targeted inhibition of PDMS polymerization. Using single channels, we first demonstrate how randomly distributed particles can be focused into the centre position of the channel in flows through low aspect ratio channels and can be effectively fractionated. As a proof of principle, continuous focusing and filtration of 10 μm particles from a suspension mixture using 4- and 16-parallel-channel devices with a single inlet and two outlets are demonstrated. A filtration efficiency of 95-97% was achieved at throughputs several orders of magnitude higher than previously shown for flows through straight channels. The scalable and low-footprint focusing device requiring neither external force fields nor mechanical parts to operate is readily applicable for high-throughput focusing and filtration applications as a stand-alone device or integrated with lab-on-a-chip systems.

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    fulltext
  • 17.
    Joensson, Haakan
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Samuels, M. L.
    Brouzes, E. R.
    Medkova, M.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Link, D. R.
    Concurrent multi-sample analysis of low expressed biomarkers on single human cells by enzymatically amplified immunodetection in droplets2008In: 12th International Conference on Miniaturized Systems for Chemistry and Life Sciences - The Proceedings of MicroTAS 2008 Conference, Chemical and Biological Microsystems Society , 2008, p. 1287-1289Conference paper (Refereed)
    Abstract [en]

    We have developed a novel microfluidic droplet based assay for analysis of low expressed cell surface proteins on individual cells at rates of hundreds of cells/s by antibody coupled enzymatic amplification in monodisperse droplets [1]. Here we expand the method to include concurrent analysis of multiple populations of single cells. We report the validation of the method by analyzing the human monocytic cell line U937 for two low expressed markers, CCR5 and CD19. Comparing our method to standard flow cytometry, we demonstrate increased peak separation, which should allow sorting by these low expressed biomarkers unavailable to flow cytometry.

  • 18.
    Jönsson, Håkan
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Droplet microfluidics for high throughput biological analysis2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Many areas of biological research increasingly perform large-scale analyses.  In genomics the entire gene repertoire of an organism is analyzed.  Proteomics attempts to understand the function and expression patterns of all proteins in a cell or organism.  Cell biologists study large numbers of single cells to understand the heterogeneity of cell populations.  In biotechnology and synthetic biology researchers search for new functional biomolecules in large libraries of biomolecular diversity e.g. for uses in medicine or bioprocessing.  More and more all of these fields employ high throughput methods to achieve the scale of analysis necessary.

    Miniaturization and parallelization provide routes towards high throughput analysis, which have proven successful for microelectronics as well as for DNA sequencing.  For the analysis of cells and biomolecules, native to an aqueous environment, miniaturization and parallelization hinges on the handling and parallel processing of very small amounts of water.  Droplet microfluidics utilizes stable picoliter (water) droplets contained in inert fluorinated oils as compartments in which to isolate and analyze cells, molecules or reactions.  These droplets can be manipulated, detected and analyzed at rates of thousands per second in microfluidic modules combining top-down microscale fabrication with the self-assembly of droplets of exact size.

    The studies constituting this thesis involve new droplet based biomolecular and single cell assays, manipulation techniques and device fabrication methods to extend the capabilities of droplet microfluidics for high throughput biological analysis.

    The first paper in the thesis describes a novel analysis method for studying the low abundant biomarkers present on the surface individual cells at resolutions not available by flow cytometry, the current gold standard of single cell analysis.  The use of a fluorescent optical dye code enabled the analysis of several single cell samples concurrently, improving throughput.

    Further a deterministic lateral displacement module, providing passive separation of droplets by size in a microfluidic circuit at more than twice higher rates than previously achievable was demonstrated.  Using this module, droplets were separated for cell occupancy based on a cell induced droplet size transformation, which couples a biological property of the droplet contents to a physical property of the droplet.  This effect, which enables passive separation of at high throughput, indicates a potential novel assay format for clone selection.

    One important feature of droplets for encapsulated single cell analysis is retention of secreted molecules providing a genotype-phenotype link.  With the objective of detecting antibody molecules secreted by hybridoma for selection, Paper III demonstrates the adaption of a homogeneous fluorescence polarization based, “mix-incubate-read”, assay for antibody detection.  In the final paper of the thesis the development of inexpensive and robust optical filters monolithically integrated in the microfluidic chip is reported. These defined filters enable integration of multiple optical filters in a polymer microfluidic device.

    Overall, droplet microfluidics combines techniques for handling and manipulating millions of discrete biocompatible picoliter compartments per hour with dedicated assays for biomolecule and single cell analysis. The scale of analysis that this enables is certain to impact life science research.

     

  • 19.
    Jönsson, Håkan N.
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Droplet microfluidics-A tool for single-cell analysis2012In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 51, no 49, p. 12176-12192Article, review/survey (Refereed)
    Abstract [en]

    Droplet microfluidics allows the isolation of single cells and reagents in monodisperse picoliter liquid capsules and manipulations at a throughput of thousands of droplets per second. These qualities allow many of the challenges in single-cell analysis to be overcome. Monodispersity enables quantitative control of solute concentrations, while encapsulation in droplets provides an isolated compartment for the single cell and its immediate environment. The high throughput allows the processing and analysis of the tens of thousands to millions of cells that must be analyzed to accurately describe a heterogeneous cell population so as to find rare cell types or access sufficient biological space to find hits in a directed evolution experiment. The low volumes of the droplets make very large screens economically viable. This Review gives an overview of the current state of single-cell analysis involving droplet microfluidics and offers examples where droplet microfluidics can further biological understanding. A one-off: Single-cell analysis is one of the most interesting applications for droplet microfluidics. Droplets provide robust compartments on the size scale of a single cell, and their ability to encapsulate and rapidly manipulate cells along with their immediate environment in monodisperse compartments allows the possibility of automation. This Review focuses on single-cell analyses and applications in drug screening and genetic and enzyme analysis.

  • 20.
    Jönsson, Håkan N.
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Droplet microfluidics: A tool for protein engineering and analysis2011In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 11, no 24, p. 4144-4147Article in journal (Refereed)
  • 21.
    Jönsson, Håkan
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Samuels, Michael L.
    Brouzes, Eric R.
    Medkova, Martina
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Link, Darren R.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Detection and Analysis of Low-Abundance Cell-Surface Biomarkers Using Enzymatic Amplification in Microfluidic Droplets2009In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 48, no 14, p. 2518-2521Article in journal (Refereed)
    Abstract [en]

    Finding the few: Cell-surface proteins are useful disease biomarkers, but current high-throughput methods are limited to detecting cells expressing more than several hundred proteins. Enzymatic amplification in microfluidic droplets (see picture) is a high-throughput method for detection and analysis of cell-surface biomarkers expressed at very low levels on individual human cells. Droplet optical labels allow concurrent analysis of several samples.

  • 22.
    Jönsson, Håkan
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Svahn Andersson, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Tröpfchen-Mikrofluidik für die Einzelzellanalyse2012In: Angewandte Chemie, ISSN 0044-8249, E-ISSN 1521-3757, Angewandte Chemie, Vol. 124, no 49, p. 12342-12359Article in journal (Refereed)
    Abstract [de]

    Die tröpfchenbasierte Mikrofluidik dient der Isolierung und Manipulation von einzelnen Zellen und Reagentien innerhalb von monodispersen, pikolitergroßen Flüssigkapseln bei einem Umsatz von tausenden Tröpfchen pro Sekunde. Diese Qualitäten machen die Tröpfchen‐Mikrofluidik geeignet für viele Anforderungen der Einzelzellanalyse. Durch die Monodispersität lässt sich die Konzentration in den Tröpfchen quantitativ einstellen. Die Tröpfchen bieten der Zelle und ihrer unmittelbaren Umgebung ein isoliertes Kompartiment, und bei einem Durchsatz von tausenden Tröpfchen pro Sekunde ist es möglich, zehntausende bis millionen verkapselte Zellen zu prozessieren. Heterogene Zellpopulationen lassen sich somit exakt beschreiben oder seltene Zellarten identifizieren. Das kleine Volumen der Tröpfchen macht auch sehr große Screenings ökonomisch machbar. Dieser Aufsatz gibt einen Überblick über den aktuellen Stand der Einzelzellanalyse durch die Tröpfchen‐Mikrofluidik und nennt Beispiele, bei denen sie biologische Vorgänge besser verstehen hilft.

  • 23.
    Jönsson, Håkan
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Deterministic lateral displacement device for droplet separation by size - Towards rapid clonal selection based on droplet shrinking2010In: 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010: Volume 2, 2010, p. 1355-1357Conference paper (Refereed)
    Abstract [en]

    We present a novel method for robust passive separation of microfluidic droplets by size using deterministic lateral displacement(DLD). We also show that droplets containing Saccharomyces Cervisiae shrink significantly during incubation while droplets containing only yeast media retain their size. We demonstrate the DLD device by sorting out shrunken yeast-cell containing droplets from a 40-fold excess of ∼33% larger yeast-cell-free droplets generated at the same time, suggesting that DLD might be used for clonal selection. The same device also separates 11 μm from 30μm droplets at a rate of 12000droplets/second, more than twofold faster than previously demonstrated passive hydrodynamic separation devices [1].

  • 24.
    Jönsson, Håkan
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Droplet size based separation by deterministic lateral displacement: separating droplets by cell-induced shrinking2011In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 11, no 7, p. 1305-1310Article in journal (Refereed)
    Abstract [en]

    We present a novel method for passive separation of microfluidic droplets by size at high throughput using deterministic lateral displacement (DLD). We also show that droplets containing Saccharomyces cerevisiae shrink significantly during incubation while droplets containing only yeast media retain or slightly increase their size. We demonstrate the DLD device by sorting out shrunken yeast-cell containing droplets from 31% larger diameter droplets which were generated at the same time containing only media, present at a >40-fold excess. This demonstrates the resolving power of droplet separation by DLD and establishes that droplets can be separated for a biological property of the droplet contents discriminated by a change of the physical properties of the droplet. Thus suggesting that this technique may be used for e.g. clonal selection. The same device also separates 11 µm from 30 µm droplets at a rate of 12000 droplets per second, more than twofold faster than previously demonstrated passive hydrodynamic separation devices.

  • 25.
    Jönsson, Håkan
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Zhang, Chi
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Sjöström, Staffan
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Microfluidic droplet based enzyme variant screening: Towards improved enzymes for industrial applications2011In: 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011, 2011, p. 179-181Conference paper (Refereed)
    Abstract [en]

    We present a microdroplet based assay for selection of efficient variants of bacterially produced amylase enzyme to improve these enzymes for industrial applications. Fluorescent analysis of α-amylase in droplets at relevant concentrations demonstrates the discrimination of wild type α-amylase at stressed and non-stressed conditions. Dielectrophoretic sorting enables enrichment of target droplets from 48% to 98.1%. Finally the viability and proliferation of Bacillus Subtilis in droplets is demonstrated. This demonstrates an enzyme analysis and screening assay in the microfluidic droplets format for selection of an industrially relevant enzyme and a basis for further enzyme selections where fluorogenic substrates are available.

  • 26.
    Jönsson, Håkan
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Zhang, Chi
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO), Proteomics.
    A homogeneous assay for biomolecule interaction analysis in droplets by flourescence polarization2010In: 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010: Volume 3, 2010, p. 1802-1804Conference paper (Refereed)
    Abstract [en]

    We present a novel homogeneous assay for detecting biomolecule interactions in microdroplets by fluorescence polarization (FP) for the first time. The FP assay allows the detection of target biomolecules directly after incubation without removing the detection reagent by separation or washing, making the assay amenable to automation. Using this assay we evaluate protein-protein and drug-DNA interactions. We detect these interactions at concentrations as low as 100nM and 69 pM respectively. This is a proof-of-concept homogeneous labeling assay in droplets for detecting bio-macromolecules.

  • 27.
    Jönsson, Håkan
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Zhang, Chi
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    A Homogeneous Assay for Protein Analysis in Droplets by Fluorescence Polarization2012In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 33, no 3, p. 436-439Article in journal (Refereed)
    Abstract [en]

    We present a novel homogeneous (mix-incubate-read) droplet microfluidic assay for specific protein detection in picoliter volumes by fluorescence polarization (FP), for the first time demonstrating the use of FP in a droplet microfluidic assay. Using an FP-based assay we detect streptavidin concentrations as low as 500?nM and demonstrate that an FP assay allows us to distinguish droplets containing 5?mu M rabbit IgG from droplets without IgG with an accuracy of 95%, levels relevant for hybridoma screening. This adds to the repertoire of droplet assay techniques a direct protein detection method which can be performed entirely inside droplets without the need for labeling of the analyte molecules.

  • 28.
    Karlsson, J. Mikael
    et al.
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Haraldsson, Tommy
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Carlborg, Carl Fredrik
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Hansson, Jonas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    van der Wijngaart, Wouter
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Fabrication and transfer of fragile 3D PDMS microstructures2012In: Journal of Micromechanics and Microengineering, ISSN 0960-1317, E-ISSN 1361-6439, Vol. 22, no 8, p. 1-9Article in journal (Refereed)
    Abstract [en]

    We present a method for PDMS microfabrication of fragile membranes and 3D fluidic networks, using a surface modified water-dissolvable release material, poly(vinyl alcohol), as a tool for handling, transfer and release of fragile polymer microstructures. The method is well suited for the fabrication of complex multilayer microfluidic devices, here shown for a PDMS device with a thin gas permeable membrane and closely spaced holes for vertical interlayer connections fabricated in a single layer. To the authors knowledge, this constitutes the most advanced PDMS fabrication method for the combination of thin, fragile structures and 3D fluidics networks, and hence a considerable step in the direction of making PDMS fabrication of complex microfluidic devices a routine endeavour.

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  • 29.
    Khorshidi, Mohammad Ali
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Vanherberghen, Bruno
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Kowalewski, Jacob M.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Garrod, Kym R.
    Lindström, Sara
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    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.
    Analysis of transient migration behavior of natural killer cells imaged in situ and in vitro2011In: Integrative Biology, ISSN 1757-9694, E-ISSN 1757-9708, Vol. 3, no 7, p. 770-778Article in journal (Refereed)
    Abstract [en]

    We present a simple method for rapid and automatic characterization of lymphocyte migration from time-lapse fluorescence microscopy data. Time-lapse imaging of natural killer (NK) cells in vitro and in situ, both showed that individual cells transiently alter their migration behavior. Typically, NK cells showed periods of high motility, interrupted by transient periods of slow migration or almost complete arrests. Analysis of in vitro data showed that these periods frequently coincided with contacts with target cells, sometimes leading to target cell lysis. However, NK cells were also commonly observed to stop independently of contact with other cells. In order to objectively characterize the migration of NK cells, we implemented a simple method to discriminate when NK cells stop or have low motilities, have periods of directed migration or undergo random movement. This was achieved using a sliding window approach and evaluating the mean squared displacement (MSD) to assess the migration coefficient and MSD curvature along trajectories from individual NK cells over time. The method presented here can be used to quickly and quantitatively assess the dynamics of individual cells as well as heterogeneity within ensembles. Furthermore, it may also be used as a tool to automatically detect transient stops due to the formation of immune synapses, cell division or cell death. We show that this could be particularly useful for analysis of in situ time-lapse fluorescence imaging data where most cells, as well as the extracellular matrix, are usually unlabelled and thus invisible.

  • 30.
    LeBlanc, Neil
    et al.
    SLU , Uppsala.
    Gantelius, Jesper
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Ståhl, Karl
    SLU, Uppsala.
    Blomberg, Jonas
    Uppsala Universitetssjukhus.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Development of a magnetic bead microarray for simultaneous and simple detection of four pestiviruses2009In: Journal of Virological Methods, ISSN 0166-0934, Vol. 155, p. 1-9Article in journal (Refereed)
    Abstract [en]

    This study reports a novel method for the rapid detection and identification of the four recognized species in the pestivirus genus of the Flaviviridae family, i.e. classical swine fever virus (CSFV), border disease virus (BDV), bovine viral diarrhoea virus type 1 (BVDV1) and type 2 (BVDV2). The analysis of pestivirus PCR products was performed on microarrays by means of magnetic bead detection. The process utilizes an oligonucleotide array, onto which 5' biotinylated PCR products were hybridized, followed by visualization with streptavidin-coated magnetic particles by the naked eye, microscope or biochip reader. The assay was tested on a collection of pestiviruses that included all four species and allowed a specific and sensitive detection. Sensitivity was compared with other post-PCR detection methods, namely gel electrophoresis and suspension microarray. The results indicate that due to its high sensitivity, specificity and simple detection procedure, the magnetic bead assay provides a powerful tool for detection and identification of viral pathogens. Considering the simplicity of the assay, the protocols for hybridization and magnetic bead detection offer an emerging application for molecular diagnoses in virology that is amenable for use in a modestly equipped laboratory.

  • 31.
    Lindstrom, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Proteomics.
    Miniaturization of biological assays: Overview on microwell devices for single-cell analyses2011In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1810, no 3, p. 308-316Article, review/survey (Refereed)
    Abstract [en]

    Background: Today, cells are commonly analyzed in ensembles, i.e. thousands of cells per sample, yielding results on the average response of the cells. However, cellular heterogeneity implies the importance of studying how individual cells respond, one by one, in order to learn more about drug targeting and cellular behavior. Scope of review: This review discusses general aspects on miniaturization of biological assays and in particular summarizes single-cell assays in microwell formats. A range of microwell-based chips are discussed with regard to their well characteristics, cell handling, choice of material etc. along with available detection systems for single-cell studies. History and trends in microsystem technology, various commonly used materials for device fabrication, and conventional methods for single-cell analysis are also discussed, before a closing section with a detailed example from our research in the field. Major conclusions:A range of miniaturized and microwell devices have shown useful for studying individual cells. General significance: In vitro assays offering low volume sampling and rapid analysis in a high-throughput manner are of great interest in a wide range of single-cell applications. Size compatibility between a cell and micron-sized tools has encouraged the field of micro- and nanotechnologies to move into areas such as life sciences and molecular biology. To test as many compounds as possible against a given amount of patient sample requires miniaturized tools where low volume sampling is sufficient for accurate results and on which a high number of experiments per cm(2) can be performed. This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine. (C) 2010 Elsevier B.V. All rights reserved.

  • 32. Lindström, S.
    et al.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Single-cell culture in microwells2012In: Single-Cell Analysis, Springer Science+Business Media B.V., 2012, Vol. 853, p. 41-52Chapter in book (Refereed)
    Abstract [en]

    In order to better understand cellular processes and behavior, a controlled way of studying high numbers of single cells and their clone formation is greatly needed. This chapter describes a microwell plate with 672 wells in a standard array/slide format, applied for single-cell culture and analysis. Single cells can be seeded into each well of the plate (1) manually or (2) automatically using a sorting flow cytometer, followed by week-long culture and detection of cell growth, protein expression, etc. The glass/silicon plate is compatible with most standard instrumentation to facilitate easy handling and enable use of the plate for single-cell analysis in most laboratory settings.

  • 33.
    Lindström, Sara
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Microwell devices for single-cell analyses2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Powerful tools for detailed cellular studies are emerging, increasing the knowledge ofthe ultimate target of all drugs: the living cell. Today, cells are commonly analyzed inensembles, i.e. thousands of cells per sample, yielding results on the average responseof the cells. However, cellular heterogeneity implies the importance of studying howindividual cells respond, one by one, in order to learn more about drug targeting andcellular behavior. In vitro assays offering low volume sampling and rapid analysis in ahigh-throughput manner are of great interest in a wide range of single-cellapplications.

    This work presents a microwell device in silicon and glass, developed using standardmicrofabrication techniques. The chip was designed to allow flow-cytometric cellsorting, a controlled way of analyzing and sorting individual cells for dynamic cultureand clone formation, previously shown in larger multiwell plates only. Dependent onthe application, minor modifications to the original device were made resulting in agroup of microwell devices suitable for various applications. Leukemic cancer cellswere analyzed with regard to their clonogenic properties and a method forinvestigation of drug response of critical importance to predict long-term clinicaloutcome, is presented. Stem cells from human and mouse were maintainedpluripotent in a screening assay, also shown useful in studies on neural differentiation.For integrated liquid handling, a fluidic system was integrated onto the chip fordirected and controlled addition of reagents in various cell-based assays. The chip wasproduced in a slide format and used as an imaging tool for low-volume sampling withthe ability to run many samples in parallel, demonstrated in a protein-binding assay fora novel bispecific affinity protein. Moving from cells and proteins into geneticanalysis, a method for screening genes from clones in a rapid manner was shown bygene amplification and mutation analysis in individual wells. In summary, a microwelldevice with associated methods were developed and applied in a range of biologicalinvestigations, particularly interesting from a cell-heterogeneity perspective.

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  • 34.
    Lindström, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    A microwell array device with integrated microfluidic components for enhanced single-cell analysis2009In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 30, no 24, p. 4166-4171Article in journal (Refereed)
    Abstract [en]

    Increasing awareness of the importance of cell heterogeneity in many biological and medical contexts is prompting increasing interest in systems that allow single-cell analysis rather than conventional bulk analysis (which provides average values for variables of interest from large numbers of cells). Recently, we presented a microwell chip for long-term, high-throughput single-cell analysis. The chip has proved to be useful for purposes such as screening individual cancer and stem cells for protein/gene markers. However, liquids in the wells can only be added or changed by manually rinsing the chip, or parts of it. This procedure has several well-known drawbacks - including risks of cross-contamination, large dead volumes and laboriousness - but there have been few previous attempts to integrate liquid rinsing/switching channels in "ready-to-use" systems for single-cell analysis. Here we present a microwell system designed (using flow simulations) for single-cell analysis with integrated microfluidic components (microchannels, magnetically driven micropumps and reservoirs) for supplying the cell culture wells with reagents, or rinsing, thus facilitating controlled, directed liquid handling. It can be used totally independently, since tubing is not essential. The practical utility of the integrated system has been demonstrated by culturing endothelial cells in the microwells, and successfully applying live-cell Calcein AM staining. Systems such as this combining high-density microwell chips with microfluidic components have great potential in numerous screening applications, such as exploring the important, but frequently undetected, heterogeneity in drug responses among individual cells.

  • 35.
    Lindström, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Eriksson, M.
    Vazin, Tandis
    KTH, School of Biotechnology (BIO), Gene Technology. National Institute of Health, United States .
    Sandberg, Julia
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Frisén, J.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    A microwell chip for parallel culture and analysis of stem cells2009In: Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Chemical and Biological Microsystems Society , 2009, p. 1309-1311Conference paper (Refereed)
    Abstract [en]

    With recent findings on the role of reprogramming factors on stem cells, in vitro screening assays for studying (de)-differentiation is of great interest. We developed a miniaturized stem cell screening chip that is easily accessible and provides means of rapidly studying thousands of individual stem/progenitor cell samples, using low reagent volumes. Results presented here include weeklong culturing and differentiation assays of mouse embryonic stem cells, mouse adult neural stem cells, and human embryonic stem cells. The possibility to maintain the cells as stem/progenitor cells over time was shown, as was isolation and clonal analysis.

  • 36.
    Lindström, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Eriksson, Malin
    Vazin, Tandis
    KTH, School of Biotechnology (BIO), Gene Technology.
    Sandberg, Julia
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Frisén, Jonas
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO).
    High-Density Microwell Chip for Culture and Analysis of Stem Cells2009In: PLos ONE, ISSN 1932-6203, Vol. 4, no 9, p. e6997-Article in journal (Refereed)
    Abstract [en]

    With recent findings on the role of reprogramming factors on stem cells, in vitro screening assays for studying (de)differentiation is of great interest. We developed a miniaturized stem cell screening chip that is easily accessible and provides means of rapidly studying thousands of individual stem/progenitor cell samples, using low reagent volumes. For example, screening of 700,000 substances would take less than two days, using this platform combined with a conventional bio-imaging system. The microwell chip has standard slide format and consists of 672 wells in total. Each well holds 500 nl, a volume small enough to drastically decrease reagent costs but large enough to allow utilization of standard laboratory equipment. Results presented here include weeklong culturing and differentiation assays of mouse embryonic stem cells, mouse adult neural stem cells, and human embryonic stem cells. The possibility to either maintain the cells as stem/progenitor cells or to study cell differentiation of stem/progenitor cells over time is demonstrated. Clonality is critical for stem cell research, and was accomplished in the microwell chips by isolation and clonal analysis of single mouse embryonic stem cells using flow cytometric cell-sorting. Protocols for practical handling of the microwell chips are presented, describing a rapid and user-friendly method for the simultaneous study of thousands of stem cell cultures in small microwells. This microwell chip has high potential for a wide range of applications, for example directed differentiation assays and screening of reprogramming factors, opening up considerable opportunities in the stem cell field.

  • 37.
    Lindström, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Hammond, Maria
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    PCR amplification and genetic analysis in a microwell cell culturing chip2009In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, p. 3465-3471Article in journal (Refereed)
    Abstract [en]

    We have previously described a microwell chip designed for high throughput, long-term single-cell culturing and clonal analysis in individual wells providing a controlled way of studying high numbers of individual adherent or non-adherent cells. Here we present a method for the genetic analysis of cells cultured on-chip by PCR and minisequencing, demonstrated using two human adherent cell lines: one wild type and one with a single-base mutation in the p53 gene. Five wild type or mutated cells were seeded per well (in a defined set of wells, each holding 500 nL of culture medium) in a 672-microwell chip. The cell chip was incubated overnight, or cultured for up to five days, depending on the desired colony size, after which the cells were lysed and subjected to PCR directly in the wells. PCR products were detected, in the wells, using a biotinylated primer and a fluorescently labelled primer, allowing the products to be captured on streptavidin-coated magnetic beads and detected by a fluorescence microscope. In addition, to enable genetic analysis by minisequencing, the double-stranded PCR products were denatured and the immobilized strands were kept in the wells by applying a magnetic field from the bottom of the wells while the wells were washed, a minisequencing reaction mixture was added, and after incubation in appropriate conditions the expected genotypes were detected in the investigated microwells, simultaneously, by an array scanner. We anticipate that the technique could be used in mutation frequency screening, providing the ability to correlate cells' proliferative heterogeneity to their genetic heterogeneity, in hundreds of samples simultaneously. The presented method of single-cell culture and DNA amplification thus offers a potentially powerful alternative to single-cell PCR, with advantageous robustness and sensitivity

  • 38.
    Lindström, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Hammond, Maria
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Gantelius, Jesper
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    PCR amplification and genetic analysis in a microwell cell cultivation chip2008In: 12th International Conference on Miniaturized Systems for Chemistry and Life Sciences - The Proceedings of MicroTAS 2008 Conference, Chemical and Biological Microsystems Society , 2008, p. 576-578Conference paper (Refereed)
    Abstract [en]

    We present a method for long-term single cell/clone cultivation followed by cell lysis, DNA amplification and detection of PCR product in a chip containing 672 individual microwells. By performing all steps on-chip in microwells, the proliferation and cell morphology of every single cell or clone can be linked to its genetic information. In this study two mammalian cell lines (mutated A431 vs. wild type U-2 OS) were used as a model system for mutation screening in the p53 gene. The presented method could improve the sensitivity in mutation frequency analysis of heterogeneous tumor samples.

  • 39.
    Lindström, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Iles, A.
    Persson, Johanna
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Hosseinkhani, H.
    Hosseinkhani, M.
    Khademhosseini, A.
    Lindström, H.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Nanoporous titania coating of microwell chips for stem cell culture and analysis2010In: Journal of Biomechanical Science and Engineering, ISSN 1880-9863, Vol. 5, no 3, p. 272-279Article in journal (Refereed)
    Abstract [en]

    Stem cell research is today an active and promising field of research. To learn more about the biology of stem cells, technical improvements are needed such as tools to study stem cells in order to characterize them further and to gain insights to the molecular regulations of their maintenance, differentiation and identification. Common procedure when studying stem cells is to coat the surface where the stem cells are to be cultured with organic materials like matri-gel, poly-L-lysine and fibronectin. The resulting coating is usually relatively fragile and it is difficult to know if the coating is evenly distributed. In addition, these forms of coatings cannot be sterilized and re-used, but must be added as an initial, time-consuming step in the daily protocol. A microwell chip with hundreds of 500 nl wells has recently been shown to be a useful tool for stem cell culturing. This platform is here modified to facilitate and improve the coating conditions for adherent cell culture. A robust and highly porous film of TiO2 is deposited in the wells prior cell seeding. TiO2 is known to be biocompatible and provides a surface that is even and well characterized, simple to produce and re-usable. Furthermore it enables the microwell chips to be stored pre-coated for longer periods of time before use. We investigated the growth of rat mesenchymal stem cells on nanoporous titania films and found that they proliferated much faster than on conventional coatings. The combination of the robust TiO2 coating of the microwell chip enables thousands of individually separated single, or clones of, stem cells to be studied simultaneously and opens up the possibility for more user-friendly cell culturing.

  • 40.
    Lindström, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Larsson, R.
    Andersson, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    High throughput single cell clone analysis2006In: Micro Total Analysis Systems - Proceedings of MicroTAS 2006 Conference: 10th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Japan Academic Association Inc , 2006, p. 410-412Conference paper (Refereed)
    Abstract [en]

    A novel microplate has been developed for high throughput single cell/clone analysis. Rapid single cell seeding using a conventional FACS into micro wells allows several thousands of single cells to be cultivated, short-term (72 h) or long-term (10-14 days), and analyzed individually. The platform requires a remarkably low number of cells, a major advantage when screening limited amounts of patient cell samples. Analysis of single cell heterogeneity and colony formation related to drug sensitivity can be accomplished in a high throughput manner.

  • 41.
    Lindström, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Larsson, Rolf
    Univ Uppsala Hosp, Dept Med Sci, Uppsala, Sweden.
    Andersson Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Towards high-throughput single cell/clone cultivation and analysis2008In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 29, p. 1219-1227Article in journal (Refereed)
    Abstract [en]

    In order to better understand cellular processes and behavior, a controlled way of studying high numbers of single cells and their clone formation is greatly needed. Numerous ways of ordering single cells into arrays have previously been described, but platforms in which each cell/clone can be addressed to an exact position in the microplate, cultivated for weeks and treated separately in a high-throughput manner have until now been missing. Here, a novel microplate developed for high-throughput single cell/clone cultivation and analysis is presented. Rapid single cell seeding into microwells, using conventional flow cytometry, allows several thousands of single cells to be cultivated, short-term (72 h) or long-term (10-14 days), and analyzed individually. By controlled sorting of individual cells to predefined locations in the microplate, analysis of single cell heterogeneity and clonogenic properties related to drug sensitivity can be accomplished. Additionally, the platform requires remarkably low number of cells, a major advantage when screening limited amounts of patient cell samples. By seeding single cells into the microplate it is possible to analyze the cells for over 14 generations, ending up with more than 10 000 cells in each well. Described here is a proof-of-concept on compartmentalization and cultivation of thousands of individual cells enabling heterogeneity analysis of various cells/clones and their response to different drugs.

  • 42. Llobera, Andreu
    et al.
    Demming, Stefanie
    Jönsson, Håkan N.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Vila-Planas, J.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Büttgenbach, Stephanus
    Monolithic PDMS passband filters for fluorescence detection2010In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 10, no 15, p. 1987-1992Article in journal (Refereed)
    Abstract [en]

    We present the fabrication and characteristics of monolithically integrated ink dyed poly(dimethylsiloxane) (PDMS) filters for optical sensing in disposable lab-on-a-chip. This represents a migration of auxillary functions onto the disposable chip with the goal of producing truly portable systems. Filters made from commercially available ink (Pelikan) directly mixed into PDMS oligomer without the use of any additional solvents were patterned with standard soft lithography technologies. Furthermore, a fabrication process based on capillary forces is presented allowing PDMS coloration of arbitrary shapes. Different filters of varying thickness fabricated using red, green and blue ink in four different concentrations were characterized. The optimal performance was found with filter thicknesses of 250 mm and ink to PDMS ratios of 0.1 (mL ink : mL PDMS oligomer) resulting in a transmittance ranging from -15.1 dB to -12.3 dB in the stopband and from -4.0 dB to -2.5 dB in the passband. Additionally, we demonstrate the robustness of this approach as the ink dyed PDMS filters do not exhibit temporal ageing due to diffusion or autofluorescence. We also show that such filters can easily be integrated in fluorescence systems, with stopbands efficient enough to allow fluorescence measurements under non-optimal conditions (broadband excitation, 180 degrees configuration). Integrated ink dyed PDMS filters add robust optical functionalities to disposable microdevices at a low cost and will enable the use of these devices for a wide range of fluorescence and absorbance based biological and chemical analysis.

  • 43. Ohashi, T.
    et al.
    Mori, K.
    Lindström, S.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Novel bioassay system for single cells2009In: Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Chemical and Biological Microsystems Society , 2009, p. 1171-1173Conference paper (Refereed)
    Abstract [en]

    In this paper, we propose a novel bioassay system consisting of an active microfluidic device integrated on a microwell plate for high throughput single-cell analysis. Firstly, a CFD (computational fluid dynamics) simulation is performed to determine flow fields in order to assess how fluid flow reaches cells cultured in the wells in the microwell plate. It is then confirmed that the cells are appropriately stained with Calcein AM by fluid flow generated by magnetically driven micropumps. The proposed bioassay system would be a powerful platform for single-cell characterization in a high throughput manner.

  • 44. Ohlander, Anna
    et al.
    Zilio, Caterina
    Hammerle, Tobias
    Zelenin, Sergey
    KTH, School of Biotechnology (BIO), Proteomics. KTH, School of Biotechnology (BIO), Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Klink, Gerhard
    Chiari, Marcella
    Bock, Karlheinz
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics. KTH, School of Biotechnology (BIO), Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Genotyping of single nucleotide polymorphisms by melting curve analysis using thin film semi-transparent heaters integrated in a lab-on-foil system2013In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 13, no 11, p. 2075-2082Article in journal (Refereed)
    Abstract [en]

    The recent technological advances in micro/nanotechnology present new opportunities to combine microfluidics with microarray technology for the development of small, sensitive, single-use, point-of-care molecular diagnostic devices. As such, the integration of microarray and plastic microfluidic systems is an attractive low-cost alternative to glass based microarray systems. This paper presents the integration of a DNA microarray and an all-polymer microfluidic foil system with integrated thin film heaters, which demonstrate DNA analysis based on melting curve analysis (MCA). A novel micro-heater concept using semi-transparent copper heaters manufactured by roll-to-roll and lift-off on polyethylene naphthalate (PEN) foil has been developed. Using a mesh structure, heater surfaces have been realized in only one single metallization step, providing more efficient and homogenous heating characteristics than conventional meander heaters. A robust DNA microarray spotting protocol was adapted on Parylene C coated heater-foils, using co-polymer poly(DMA-NAS-MAPS) to enable covalent immobilization of DNA. The heaters were integrated in a microfluidic channel using lamination foils and MCA of the spotted DNA duplexes showed single based discrimination of mismatched over matched target DNA-probes. Finally, as a proof of principle, we perform MCA on PCR products to detect the Leu7Pro polymorphism of the neutropeptide Y related to increased risk of Type II diabetes, BMI and depression.

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  • 45.
    Ramachandraiah, Harisha
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Amasia, Mary
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Cole, J.
    Sheard, P.
    Pickhaver, S.
    Walker, C.
    Lione, R.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Centrifugal microfluidic system for rapid, low-cost HIV diagnosis: CD4+ T-cell counting using an integrated DVD platform2012In: Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, Chemical and Biological Microsystems Society , 2012, p. 1942-1944Conference paper (Refereed)
    Abstract [en]

    HIV is a pandemic that currently threatens over 33 million lives worldwide and HIV/AIDS remains one of the major causes of death globally. The continued monitoring of the CD4+ T-lymphocytes count in HIV patients is necessary for proper treatment, although this testing is too expensive and complex for limited resource settings. We report on a novel integrated centrifugal (CD) microfluidic system for rapid and low-cost HIV diagnosis through automated counting of CD4+ T-cells for point-of-care applications. We demonstrate the integrated T-cell immunocapture and detection mechanism using a novel system comprised of a modified commercial DVD drive and polymer disc.

  • 46.
    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)
  • 47.
    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)
  • 48.
    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)
  • 49.
    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.

  • 50. Shirai, K.
    et al.
    Renberg, Björn
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Sato, K.
    Mawatari, K.
    Kitamori, T.
    Graft linker immobilization for spatial control of antibody immobilization inside fused microchips2009In: Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Chemical and Biological Microsystems Society , 2009, p. 1566-1568Conference paper (Refereed)
    Abstract [en]

    A new method for antibody immobilization to the inside of fused silica microchips coated with a protein repelling polymer (2-methacryloyloxyethyl phosphorylcholine polymer, MPC polymer) is described. Antibody patterning is achieved by using a new photoreactive linker which is composed of three-parts: a carboxyl group (-COOH), a hydrophilic PEG spacer and a photoreactive benzophenone. The linker is grafted with UV which introduces free carboxyl groups to the microchannel surface. Antibodies are immobilized to these areas, by using amide bonding between protein -NH2's and linker's -COOH. Patterned anti-BSA antibodies retained functionality and were capable of antigen specific capture.

12 1 - 50 of 63
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