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  • 1.
    Afrasiabi, Roodabeh
    et al.
    KTH, Skolan för informations- och kommunikationsteknik (ICT), Material- och nanofysik.
    Söderberg, Lovisa M.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Jönsson, Håkan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Björk, Per
    Acreo Swedish ICT AB, SE-164 40 Kista, Sweden.
    Svahn Andersson, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Linnros, Jan
    KTH, Skolan för informations- och kommunikationsteknik (ICT), Material- och nanofysik.
    Integration of a Droplet-Based Microfluidic System and Silicon Nanoribbon FET Sensor2016Ingår i: Micromachines, ISSN 2072-666X, E-ISSN 2072-666X, Vol. 7, nr 8Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We present a novel microfluidic system that integrates droplet microfluidics with a silicon nanoribbon field-effect transistor (SiNR FET), and utilize this integrated system to sense differences in pH. The device allows for selective droplet transfer to a continuous water phase, actuated by dielectrophoresis, and subsequent detection of the pH level in the retrieved droplets by SiNR FETs on an electrical sensor chip. The integrated microfluidic system demonstrates a label-free detection method for droplet microfluidics, presenting an alternative to optical fluorescence detection. In this work, we were able to differentiate between droplet trains of one pH-unit difference. The pH-based detection method in our integrated system has the potential to be utilized in the detection of biochemical reactions that induce a pH-shift in the droplets.

  • 2.
    Bai, Yunpeng
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Weibull, Emilie
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Jönsson, Håkan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Andersson Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Interfacing picoliter droplet microfluidics with addressable microliter compartments using fluorescence activated cell sorting2014Ingår i: Sensors and actuators. B, Chemical, ISSN 0925-4005, E-ISSN 1873-3077, Vol. 194, s. 249-254Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Droplet microfluidic platforms have, while enabling high-throughput manipulations and the assaying of single cell scale compartments, been lacking interfacing to allow macro scale access to the output from droplet microfluidic operations. Here, we present a simple and high-throughput method for individually directing cell containing droplets to an addressable and macro scale accessible microwell slide for downstream analysis. Picoliter aqueous droplets containing low gelling point agarose and eGFP expressing Escherichia coli (E. coli) are created in a microfluidic device, solidified to agarose beads and transferred into an aqueous buffer. A Fluorescence activated cell sorter (FACS) is used to sort agarose beads containing cells into microwells in which the growth and expansion of cell colonies is monitored. We demonstrate fast sorting and high accuracy positioning of sorted 15 μm gelled droplet agarose beads into microwells (14 × 48) on a 25 mm × 75 mm microscope slide format using a FACS with a 100 μm nozzle and an xy-stage. The interfacing method presented here enables the products of high-throughput or single cell scale droplet microfluidics assays to be output to a wide range of microtiter plate formats familiar to biological researchers lowering the barriers for utilization of these microfluidic platforms.

  • 3.
    Björk, S. M.
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. Novo Nordisk Foundation Center for Biosustainability.
    Sjöström, S. L.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. Novo Nordisk Foundation Center for Biosustainability.
    Andersson-Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. Novo Nordisk Foundation Center for Biosustainability.
    Jönsson, Håkan N.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. Novo Nordisk Foundation Center for Biosustainability.
    Tuning microfluidic cell culture conditions for droplet based screening by metabolite profiling2015Ingår i: MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Chemical and Biological Microsystems Society , 2015, s. 1377-1379Konferensbidrag (Refereegranskat)
    Abstract [en]

    We investigate the impact of droplet culture conditions on cell metabolic state by determining key metabolite concentrations in S. cerevisiae cultures in different microfluidic droplet culture formats. Control of culture conditions is critical for single cell screening in droplets, as cell metabolic state directly affects production yields in cell factories. Metabolite profiling provides a more nuanced estimate of cell state compared to proliferation studies alone. We show that the choice of droplet incubation format has an impact on cell proliferation and metabolite production. Furthermore, we engineered a new better oxygenated droplet incubation format, with retained droplet stability and size.

  • 4.
    Björk, Sara
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.
    Jönsson, Håkan
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.
    Microfluidics for cell factory and bioprocess development2019Ingår i: Current Opinion in Biotechnology, ISSN 0958-1669, E-ISSN 1879-0429, Vol. 55, s. 95-102Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bioindustry is expanding to an increasing variety of food, chemical and pharmaceutical products, each requiring rapid development of a dedicated cell factory and bioprocess. Microfluidic tools are, together with tools from synthetic biology and metabolic modeling, being employed in cell factory and bioprocess development to speed up development and address new products. Recent examples of microfluidics for bioprocess development range from integrated devices for DNA assembly and transformation, to high throughput screening of cell factory libraries, and micron scale bioreactors for process optimization. These improvements act to improve the biotechnological engineering cycle with tools for building, testing and evaluating cell factories and bioprocesses by increasing throughput, parallelization and automation.

  • 5.
    Björk, Sara M.
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.
    Sjostrom, Staffan L.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Andersson-Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.
    Jönsson, Håkan N.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.
    Metabolite profiling of microfluidic cell culture conditions for droplet based screening2015Ingår i: Biomicrofluidics, ISSN 1932-1058, E-ISSN 1932-1058, Vol. 9, nr 4, artikel-id 044128Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We investigate the impact of droplet culture conditions on cell metabolic state by determining key metabolite concentrations in S. cerevisiae cultures in different microfluidic droplet culture formats. Control of culture conditions is critical for single cell/clone screening in droplets, such as directed evolution of yeast, as cell metabolic state directly affects production yields from cell factories. Here, we analyze glucose, pyruvate, ethanol, and glycerol, central metabolites in yeast glucose dissimilation to establish culture formats for screening of respiring as well as fermenting yeast. Metabolite profiling provides a more nuanced estimate of cell state compared to proliferation studies alone. We show that the choice of droplet incubation format impacts cell proliferation and metabolite production. The standard syringe incubation of droplets exhibited metabolite profiles similar to oxygen limited cultures, whereas the metabolite profiles of cells cultured in the alternative wide tube droplet incubation format resemble those from aerobic culture. Furthermore, we demonstrate retained droplet stability and size in the new better oxygenated droplet incubation format.

  • 6.
    Björk, Sara
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.
    Schappert, Martin
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.
    Jönsson, Håkan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.
    Droplet microfluidic microcolony analysis of triacylglycerol yields in S. cerevisiae for high throughput screeningManuskript (preprint) (Övrigt vetenskapligt)
  • 7.
    Björk, Sara
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Shabestary, Kiyan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Yao, Lun
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Ljungqvist, Emil
    Jönsson, Håkan
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hudson, Elton P.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Droplet microfluidic screening of a Synechocystis sp. CRISPRi library based on L-lactate productionManuskript (preprint) (Övrigt vetenskapligt)
  • 8.
    Björk, Sara
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Sjöström, Staffan L.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Andersson Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Jönsson, Håkan N.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Controlling cell metabolic state in droplet microfluidicsManuskript (preprint) (Övrigt vetenskapligt)
  • 9. Fornell, A.
    et al.
    Jonsson, Håkan N.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Nilsson, J.
    Tenje, M.
    Acoustic focusing of microparticles in two-phase systems - Towards cell enrichment or medium exchange in droplets2015Ingår i: MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Chemical and Biological Microsystems Society , 2015, s. 1026-1028Konferensbidrag (Refereegranskat)
    Abstract [en]

    We present a method to first laterally position microparticles inside droplets by acoustic forces and then split the droplet into three daughter droplets to achieve a 2+fold enrichment of microparticles inside the center droplet. We show that acoustic forces can be applied to both manipulate polystyrene beads (5 μm) and red blood cells inside droplets. The presented technology opens up for development of droplet operations used for medium exchange and particle concentration in droplet-based cell assays.

  • 10. Fornell, Anna
    et al.
    Nilsson, Johan
    Jonsson, Linus
    Periyannan Rajeswari, Prem Kumar
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.
    Jönsson, Håkan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.
    Tenje, Maria
    Particle enrichment in droplet acoustofluidics2016Ingår i: Micronano System Workshop (MSW 2016), Lund, Sweden, May 17-18 2016, 2016Konferensbidrag (Refereegranskat)
  • 11. Fornell, Anna
    et al.
    Nilsson, Johan
    Jonsson, Linus
    Periyannan Rajeswari, Prem Kumar
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Jönsson, Håkan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Tenje, Maria
    Particle enrichment in two-phase microfluidic systems using acoustophoresis2016Ingår i: Acoustofluidics 2016, Kongens Lyngby, Denmark, September 22-23 2016, 2016Konferensbidrag (Refereegranskat)
  • 12. Fornell, Anna
    et al.
    Nilsson, Johan
    Jonsson, Linus
    Rajeswari, Prem Kumar Periyannan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Jönsson, Håkan N.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Tenje, Maria
    Controlled Lateral Positioning of Microparticles Inside Droplets Using Acoustophoresis2015Ingår i: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 87, nr 20, s. 10521-10526Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 13.
    Hammar, Petter
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Angermayr, S. Andreas
    Sjöström, Staffan L.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    van der Meer, Josefin
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hellingwerf, Klaas J.
    Hudson, Elton P.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Jönsson, Håkan N.
    Novo Nordisk Foundation Center for Biosustainability.
    Single-cell screening of photosynthetic growth and lactate production by cyanobacteria2015Ingår i: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 8, artikel-id 193Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Photosynthetic cyanobacteria are attractive for a range of biotechnological applications including biofuel production. However, due to slow growth, screening of mutant libraries using microtiter plates is not feasible. Results: We present a method for high-throughput, single-cell analysis and sorting of genetically engineered l-lactate-producing strains of Synechocystis sp. PCC6803. A microfluidic device is used to encapsulate single cells in picoliter droplets, assay the droplets for L-lactate production, and sort strains with high productivity. We demonstrate the separation of low- and high-producing reference strains, as well as enrichment of a more productive L-lactate-synthesizing population after UV-induced mutagenesis. The droplet platform also revealed population heterogeneity in photosynthetic growth and lactate production, as well as the presence of metabolically stalled cells. Conclusions: The workflow will facilitate metabolic engineering and directed evolution studies and will be useful in studies of cyanobacteria biochemistry and physiology.

  • 14.
    Hammar, Petter
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Sjöström, Staffan L.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Angermayr, Andreas
    Hellingwerf, Klaas J.
    Hudson, Paul
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Jönsson, Håkan N.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Single-cell screening of secreted lactate production in cyanobacteriaManuskript (preprint) (Övrigt vetenskapligt)
  • 15. Hammond, Maria
    et al.
    Homa, Felix
    Andersson-Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Ettema, Thijs J. G.
    Jönsson, Håkan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.
    Picodroplet partitioned whole genome amplification of low biomass samples preserves genomic diversity for metagenomic analysis2016Ingår i: Microbiome, ISSN 0026-2633, E-ISSN 2049-2618, Vol. 4, artikel-id 52Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Whole genome amplification (WGA) is a challenging, key step in metagenomic studies of samples containing minute amounts of DNA, such as samples from low biomass environments. It is well known that multiple displacement amplification (MDA), the most commonly used WGA method for microbial samples, skews the genomic representation in the sample. We have combined MDA with droplet microfluidics to perform the reaction in a homogeneous emulsion. Each droplet in this emulsion can be considered an individual reaction chamber, allowing partitioning of the MDA reaction into millions of parallel reactions with only one or very few template molecules per droplet. Results: As a proof-of-concept, we amplified genomic DNA from a synthetic metagenome by MDA either in one bulk reaction or in emulsion and found that after sequencing, the species distribution was better preserved and the coverage depth was more evenly distributed across the genomes when the MDA reaction had been performed in emulsion. Conclusions: Partitioning MDA reactions into millions of reactions by droplet microfluidics is a straightforward way to improve the uniformity of MDA reactions for amplifying complex samples with limited amounts of DNA.

  • 16. Huang, M.
    et al.
    Jönsson, Håkan
    Novo Nordisk Foundation Center for Biosustainability.
    Nielsen, J.
    High-throughput microfluidics for the screening of yeast libraries2018Ingår i: Synthetic Metabolic Pathways: Methods and Protocols, Humana Press, 2018, Vol. 1671, s. 307-317Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    Cell factory development is critically important for efficient biological production of chemicals, biofuels, and pharmaceuticals. Many rounds of the Design–Build–Test–Learn cycles may be required before an engineered strain meeting specific metrics required for industrial application. The bioindustry prefer products in secreted form (secreted products or extracellular metabolites) as it can lower the cost of downstream processing, reduce metabolic burden to cell hosts, and allow necessary modification on the final products, such as biopharmaceuticals. Yet, products in secreted form result in the disconnection of phenotype from genotype, which may have limited throughput in the Test step for identification of desired variants from large libraries of mutant strains. In droplet microfluidic screening, single cells are encapsulated in individual droplet and enable high-throughput processing and sorting of single cells or clones. Encapsulation in droplets allows this technology to overcome the throughput limitations present in traditional methods for screening by extracellular phenotypes. In this chapter, we describe a protocol/guideline for high-throughput droplet microfluidics screening of yeast libraries for higher protein secretion. This protocol can be adapted to screening by a range of other extracellular products from yeast or other hosts.

  • 17. Huang, Mingtao
    et al.
    Bai, Yunpeng
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. East China University of Science and Technology, China.
    Sjöström, Staffan L.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hallström, Björn M.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Liu, Zihe
    Petranovic, Dina
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. Technical University of Denmark, Denmark .
    Jönsson, Håkan N.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Andersson Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Nielsen, Jens
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. Chalmers University of Technology, Sweden; Technical University of Denmark, Denmark.
    Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast2015Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, nr 34, s. E4689-E4696Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used highthroughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant α-amylase. Efficient secretion was genetically stable in the selected clones. We performed wholegenome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.

  • 18.
    Joensson, Haakan
    et al.
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101).
    Samuels, M. L.
    Brouzes, E. R.
    Medkova, M.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Andersson Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101).
    Link, D. R.
    Concurrent multi-sample analysis of low expressed biomarkers on single human cells by enzymatically amplified immunodetection in droplets2008Ingår i: 12th International Conference on Miniaturized Systems for Chemistry and Life Sciences - The Proceedings of MicroTAS 2008 Conference, Chemical and Biological Microsystems Society , 2008, s. 1287-1289Konferensbidrag (Refereegranskat)
    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.

  • 19.
    Jönsson, Håkan
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi.
    Droplet microfluidics for high throughput biological analysis2011Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    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.

     

  • 20.
    Jönsson, Håkan N.
    et al.
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101).
    Andersson Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101).
    Droplet microfluidics-A tool for single-cell analysis2012Ingår i: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 51, nr 49, s. 12176-12192Artikel, forskningsöversikt (Refereegranskat)
    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.

  • 21.
    Jönsson, Håkan N.
    et al.
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101).
    Andersson-Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101).
    Droplet microfluidics: A tool for protein engineering and analysis2011Ingår i: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 11, nr 24, s. 4144-4147Artikel i tidskrift (Refereegranskat)
  • 22.
    Jönsson, Håkan
    et al.
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101).
    Samuels, Michael L.
    Brouzes, Eric R.
    Medkova, Martina
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Link, Darren R.
    Andersson-Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101).
    Detection and Analysis of Low-Abundance Cell-Surface Biomarkers Using Enzymatic Amplification in Microfluidic Droplets2009Ingår i: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 48, nr 14, s. 2518-2521Artikel i tidskrift (Refereegranskat)
    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.

  • 23.
    Jönsson, Håkan
    et al.
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101).
    Svahn Andersson, Helene
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101).
    Tröpfchen-Mikrofluidik für die Einzelzellanalyse2012Ingår i: Angewandte Chemie, ISSN 0044-8249, E-ISSN 1521-3757, Angewandte Chemie, Vol. 124, nr 49, s. 12342-12359Artikel i tidskrift (Refereegranskat)
    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.

  • 24.
    Jönsson, Håkan
    et al.
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Andersson-Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101).
    Deterministic lateral displacement device for droplet separation by size - Towards rapid clonal selection based on droplet shrinking2010Ingår i: 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010: Volume 2, 2010, s. 1355-1357Konferensbidrag (Refereegranskat)
    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].

  • 25.
    Jönsson, Håkan
    et al.
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Andersson-Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi.
    Droplet size based separation by deterministic lateral displacement: separating droplets by cell-induced shrinking2011Ingår i: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 11, nr 7, s. 1305-1310Artikel i tidskrift (Refereegranskat)
    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.

  • 26.
    Jönsson, Håkan
    et al.
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101).
    Zhang, Chi
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101).
    Sjöström, Staffan
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101).
    Andersson-Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101).
    Microfluidic droplet based enzyme variant screening: Towards improved enzymes for industrial applications2011Ingår i: 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011, 2011, s. 179-181Konferensbidrag (Refereegranskat)
    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.

  • 27.
    Jönsson, Håkan
    et al.
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101).
    Zhang, Chi
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Andersson Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    A homogeneous assay for biomolecule interaction analysis in droplets by flourescence polarization2010Ingår i: 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010: Volume 3, 2010, s. 1802-1804Konferensbidrag (Refereegranskat)
    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.

  • 28.
    Jönsson, Håkan
    et al.
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101).
    Zhang, Chi
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Andersson-Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101).
    A Homogeneous Assay for Protein Analysis in Droplets by Fluorescence Polarization2012Ingår i: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 33, nr 3, s. 436-439Artikel i tidskrift (Refereegranskat)
    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.

  • 29.
    Khorshidi, Mohammad Ali
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Periyannan Rajeswari, Prem Kumar
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Wahlby, C.
    Jönsson, Håkan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Andersson Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Dynamic behavior analysis of single cells using droplet microfluidics2013Ingår i: 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013, 2013, Vol. 3, s. 1674-1676Konferensbidrag (Refereegranskat)
    Abstract [en]

    We present a droplet microfluidic platform to automatically track and characterize the behavior of single cells over time. Time series analysis of single cells was enabled by encapsulating the cells in microdroplets followed by trapping microdroplets in static array of microwells that were fabricated on chip to make the droplets addressable by their position and imaging them over time. In this paper, we demonstrate the potential of automated time-lapse imaging and image analysis approach in droplet microfluidics by studying the viability of large number of single cells over time.

  • 30.
    Khorshidi, Mohammad Ali
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Periyannan Rajeswari, Prem Kumar
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Wählby, Carolina
    Jönsson, Håkan N.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Andersson Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Automated analysis of dynamic behavior of single cells in picoliter droplets2014Ingår i: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 14, nr 5, s. 931-937Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We present a droplet-based microfluidic platform to automatically track and characterize the behavior of single cells over time. This high-throughput assay allows encapsulation of single cells in micro-droplets and traps intact droplets in arrays of miniature wells on a PDMS-glass chip. Automated time-lapse fluorescence imaging and image analysis of the incubated droplets on the chip allows the determination of the viability of individual cells over time. In order to automatically track the droplets containing cells, we developed a simple method based on circular Hough transform to identify droplets in images and quantify the number of live and dead cells in each droplet. Here, we studied the viability of several hundred single isolated HEK293T cells over time and demonstrated a high survival rate of the encapsulated cells for up to 11 hours. The presented platform has a wide range of potential applications for single cell analysis, e.g. monitoring heterogeneity of drug action over time and rapidly assessing the transient behavior of single cells under various conditions and treatments in vitro.

  • 31.
    Langer, Krzysztof
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Jönsson, Håkan
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Rapid production and recovery of cell spheroids by automated droplet microfluidics2019Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Droplet microfluidics enables high throughput cell processing, analysis and screening by miniaturizing the reaction vessels to nano- or pico-liter water-in oil droplets, but like many other microfluidic formats, droplet microfluidics have not been interfaced with or automated by laboratory robotics. Here we demonstrate automation of droplet microfluidics based on an inexpensive liquid handling robot for the automated production of human scaffold-free cell spheroids, using pipette actuation and interfacing the pipetting tip with a droplet generating microfluidic chip. In this chip we produce highly mono-disperse 290μm droplets with diameter CV of 1.7%. By encapsulating cells in these droplets, we produce cell spheroids in droplets and recover them to standard formats at a throughput of 85000 spheroids per microfluidic circuit per hour. The viability of the cells in spheroids remains high after recovery only decreased by 4% starting from 96% after 16 hours incubation in nanoliter droplets. Scaffold-free cell spheroids and 3D tissue constructs recapitulate many aspects of functional human tissue more accurately than 2D or single cell cultures, but assembly methods for spheroids, e.g. hanging drop micro-plates, has had limited throughput. The increased throughput and decreased cost of our method enables spheroid production at the scale needed for lead discovery drug screening and approaches the cost where these micro tissues could be used as building blocks for organ scale regenerative medicine.

  • 32.
    Langer, Krzysztof
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.
    Jönsson, Håkan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.
    Rapid production and recovery of cell spheroids by automated droplet microfluidics2019Ingår i: bioRxivArtikel i tidskrift (Refereegranskat)
    Abstract [en]

    Droplet microfluidics enables high throughput cell processing, analysis and screening by miniaturizing the reaction vessels to nano- or pico-liter water-in oil droplets, but like many other microfluidic formats, droplet microfluidics have not been interfaced with or automated by laboratory robotics. Here we demonstrate automation of droplet microfluidics based on an inexpensive liquid handling robot for the automated production of human scaffold-free cell spheroids, using pipette actuation and interfacing the pipetting tip with a droplet generating microfluidic chip. In this chip we produce highly mono-disperse 290μm droplets with diameter CV of 1.7%. By encapsulating cells in these droplets, we produce cell spheroids in droplets and recover them to standard formats at a throughput of 85000 spheroids per microfluidic circuit per hour. The viability of the cells in spheroids remains high after recovery only decreased by 4% starting from 96% after 16 hours incubation in nanoliter droplets. Scaffold-free cell spheroids and 3D tissue constructs recapitulate many aspects of functional human tissue more accurately than 2D or single cell cultures, but assembly methods for spheroids, e.g. hanging drop micro-plates, has had limited throughput. The increased throughput and decreased cost of our method enables spheroid production at the scale needed for lead discovery drug screening and approaches the cost where these micro tissues could be used as building blocks for organ scale regenerative medicine.

  • 33.
    Langer, Krzysztof
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Jönsson, Håkan
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi. KTH, Novo Nordisk Fdn Ctr Biosustainabil, Stockholm, Sweden.
    Rapid Production and Recovery of Cell Spheroids by Automated Droplet Microfluidics2019Ingår i: SLAS TECHNOLOGY, ISSN 2472-6303, artikel-id UNSP 2472630319877376Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The future of the life sciences is linked to automation and microfluidics. As robots start working side by side with scientists, robotic automation of microfluidics in general, and droplet microfluidics in particular, will significantly extend and accelerate the life sciences. Here, we demonstrate the automation of droplet microfluidics using an inexpensive liquid-handling robot to produce human scaffold-free cell spheroids at high throughput. We use pipette actuation and interface the pipetting tip with a droplet-generating microfluidic device. In this device, we produce highly monodisperse droplets with a diameter coefficient of variation (CV) lower than 2%. By encapsulating cells in these droplets, we produce cell spheroids in droplets and recover them to standard labware containers at a throughput of 85,000 spheroids per microfluidic circuit per hour. The viability of the cells in spheroids remains high throughout the process and decreases by >10% (depending on the cell line used) after a 16 h incubation period in nanoliter droplets and automated recovery. Scaffold-free cell spheroids and 3D tissue constructs recapitulate many aspects of functional human tissue more accurately than 2D or single-cell cultures, but assembly methods for spheroids (e.g., hanging drop microplates) have limited throughput. The increased throughput and decreased cost of our method enable spheroid production at the scale needed for lead discovery drug screening, and approach the cost at which these microtissues could be used as building blocks for organ-scale regenerative medicine.

  • 34. Llobera, Andreu
    et al.
    Demming, Stefanie
    Jönsson, Håkan N.
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi.
    Vila-Planas, J.
    Andersson-Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi.
    Büttgenbach, Stephanus
    Monolithic PDMS passband filters for fluorescence detection2010Ingår i: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 10, nr 15, s. 1987-1992Artikel i tidskrift (Refereegranskat)
    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.

  • 35.
    Periyannan Rajeswari, Prem Kumar
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Jönsson, Håkan N
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Andersson Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Droplet size influences division of mammalian cell factories in droplet microfluidic cultivation2016Ingår i: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The potential of using droplet microfluidics for screening mammalian cell factories has been limited by the difficulty in achieving continuous cell division during cultivation in droplets. Here, we report the influence of droplet size on mammalian cell division and viability during cultivation in droplets. Chinese Hamster Ovary (CHO) cells, the most widely used mammalian host cells for biopharmaceuticals production were encapsulated and cultivated in 33, 180 and 320 pL droplets for 3 days. Periodic monitoring of the droplets during incubation showed that the cell divisions in 33 pL droplets stopped after 24 h, whereas continuous cell division was observed in 180 and 320 pL droplets for 72 h. The viability of the cells cultivated in the 33 pL droplets also dropped to about 50% in 72 h. In contrast, the viability of the cells in the larger droplets was above 90% even after 72 h of cultivation, making them a more suitable droplet size for 72-h cultivation. This study shows a direct correlation of microfluidic droplet size to the division and viability of mammalian cells. This highlights the importance of selecting suitable droplet size for mammalian cell factory screening assays.

  • 36.
    Periyannan Rajeswari, Prem Kumar
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Soderberg, Lovisa M
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Andersson Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Jönsson, Håkan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Color-coded bead based readout from droplet PCR for the detection of pathogen biomarkersManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    We present a workflow using fluorescent color-coded Luminex beads to detect the

    outcome of droplet PCR assay. The assay was performed to detect three important

    poultry pathogens: avian influenza, infectious laryngotracheitis virus and

    campylobacter. Droplet-based TaqMan PCR has been commonly used for detection

    of rare and significant biomarkers in clinical samples. However, the spectral overlap

    of fluorescent TaqMan probes limits the detection to 5 different targets in a single

    assay. The color codes of the Luminex detection beads allowed accurate classification

    of the different bead sets used in this assay concurrently. The target-specific capture

    probes coupled to distinct bead sets enabled capture and detection of target DNA in

    the droplet. The capture assay detected target DNA of all three poultry pathogens with

    high specificity, from samples with average target concentration of 1 template per

    droplet. This workflow demonstrates that the detection panel of droplet PCR assay

    can be increased to potentially detect multiple targets in a sample by utilizing the

    scalability offered by the color-coded detection beads.

  • 37. Siedler, S.
    et al.
    Khatri, N. K.
    KTH.
    Zsohár, A.
    Kjærbølling, I.
    Vogt, M.
    Hammar, P.
    KTH.
    Stahlhut, S. G.
    Marienhagen, J.
    Sommer, M. O. A.
    Jönsson, Håkan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    High throughput droplet sorting of yeast for p-Coumaric acid production detected by co-encapsulated E. coli biosensor bacteria2016Ingår i: 20th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2016, Chemical and Biological Microsystems Society , 2016, s. 551-552Konferensbidrag (Refereegranskat)
    Abstract [en]

    We present the optimization of E. coli biosensor bacteria and demonstrate their use to detect and sort co-encapsulated S. cerevisiae yeast cells by their p-Coumaric acid (pCA) production in picoliter microfluidic droplets at high throughput. This strategy allows us to enrich pCA producing cell factories by the cell permeable product pCA using a biosensor in a separate cell.

  • 38. Siedler, S.
    et al.
    Khatri, Narendar K.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. Technical University of Denmark.
    Zsohár, A.
    Kjærbølling, I.
    Vogt, M.
    Hammar, Petter
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Nielsen, C. F.
    Marienhagen, J.
    Sommer, M. O. A.
    Jönsson, Håkan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. Technical University of Denmark.
    Development of a Bacterial Biosensor for Rapid Screening of Yeast p-Coumaric Acid Production2017Ingår i: ACS Synthetic Biology, E-ISSN 2161-5063, Vol. 6, nr 10, s. 1860-1869Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Transcription factor-based biosensors are used to identify producer strains, a critical bottleneck in cell factory engineering. Here, we address two challenges with this methodology: transplantation of heterologous transcriptional regulators into new hosts to generate functional biosensors and biosensing of the extracellular product concentration that accurately reflects the effective cell factory production capacity. We describe the effects of different translation initiation rates on the dynamic range of a p-coumaric acid biosensor based on the Bacillus subtilis transcriptional repressor PadR by varying its ribosomal binding site. Furthermore, we demonstrate the functionality of this p-coumaric acid biosensor in Escherichia coli and Corynebacterium glutamicum. Finally, we encapsulate yeast p-coumaric acid-producing cells with E. coli-biosensing cells in picoliter droplets and, in a microfluidic device, rapidly sort droplets containing yeast cells producing high amounts of extracellular p-coumaric acid using the fluorescent E. coli biosensor signal. As additional biosensors become available, such approaches will find broad applications for screening of an extracellular product.

  • 39.
    Sjöström, Staffan
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Huang, Mingtao
    Nielsen, Jens
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Jönsson, Håkan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Andersson Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Micro-droplet based directed evolution outperforms conventional laboratory evolution2014Ingår i: 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014, Chemical and Biological Microsystems Society , 2014, s. 169-171Konferensbidrag (Refereegranskat)
    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.

  • 40.
    Sjöström, Staffan L.
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Bai, Yunpeng
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Huang, Mingtao
    Liu, Zihe
    Nielsen, Jens
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Jönsson, Håkan N.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Andersson Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    High-throughput screening for industrial enzyme production hosts by droplet microfluidics2014Ingår i: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 14, nr 4, s. 806-813Artikel i tidskrift (Refereegranskat)
    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.

  • 41.
    Sjöström, Staffan L.
    et al.
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Jönsson, Håkan N.
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Andersson Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Nanobioteknologi (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Multiplex analysis of enzyme kinetics and inhibition by droplet microfluidics using picoinjectors2013Ingår i: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 13, nr 9, s. 1754-1761Artikel i tidskrift (Refereegranskat)
    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.

  • 42.
    Sjöström, Staffan L.
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Jönsson, Håkan N.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Andersson Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    High-­throughput screening for improved enzymes in environments lethal to host cellsManuskript (preprint) (Övrigt vetenskapligt)
  • 43.
    Sjöström, Staffan L.
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Jönsson, Håkan N.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Svahn, Helene Andersson
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Multiplex analysis of enzyme kinetics and inhibition by droplet microfluidics using picoinjectors2012Ingår i: Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, 2012, s. 172-174Konferensbidrag (Refereegranskat)
    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.

  • 44.
    Söderberg, Lovisa
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Fonseca, Pedro
    Karolinska Intitutet, Department of Oncology and Pathology.
    Panaretakis, Theocharis
    Karolinska Intitutet, Department of Oncology and Pathology.
    Jönsson, Håkan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Detection of single exosomes in microfluidic droplets by RT-PCR amplification of 18S RNA contentManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    We present a workflow for reverse transcription-PCR (RT-PCR) in microfluidic droplets to identify exosomes based on their RNA content. Available techniques for exosome detection have been limited to size or surface markers which limit their diagnostic capabilities. Exosome detection based on RNA content could be developed to be used as a diagnostic, prognostic or predictive tool for cancer based on specific RNA biomarkers in liquid biopsies. In this manuscript we demonstrate a high throughput method for the amplification of exosome derived 18S RNA in microfluidic droplets. Automated image analysis using open source software was applied to distinguish and count PCR-positive droplets with fluorescent intensity over a set threshold. We benchmark our workflow against picoliter scale RT-PCR on serially diluted exosome samples and demonstrate the ability of the droplet based workflow to correctly rank exosome samples based on exosome concentration.  This represents a key step towards a quantitative analysis of exosomal RNA content and the sorting of single exosomes by their RNA content.

  • 45.
    Söderberg, Lovisa
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Jönsson, Håkan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Andersson Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Parallel cDNA synthesis from thousands of individually encapsulated cancer cells: Towards large scale single cell gene expression analysis2013Ingår i: 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013, 2013, Vol. 3, s. 1737-1739Konferensbidrag (Refereegranskat)
    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.

  • 46.
    Volk, Anna-Luisa
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Hansen, Henning G.
    Lundqvist, Magnus
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Hammar, Petter
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Bai, Yunpeng
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Kol, Stefan
    Kildegaard, Helene F.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. Technical University of Denmark, Denmark.
    Joensson, Haakan N.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Droplet microfluidics and split-GFP complementation enable selection of Chinese hamster ovary cells with high specific productivity of therapeutic glycoproteinsManuskript (preprint) (Övrigt vetenskapligt)
  • 47. Wang, Guokun
    et al.
    Björk, Sara
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Huang, Mingtao
    Liu, Quanli
    Campbell, Kate
    Nielsen, Jens
    Jönsson, Håkan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Petranovic, Dina
    RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production in Saccharomyces cerevisiae2019Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 116, nr 19, s. 9324-9332Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The cellular machinery that supports protein synthesis and secretion lies at the foundation of cell factory-centered protein production. Due to the complexity of such cellular machinery, the challenge in generating a superior cell factory is to fully exploit the production potential by finding beneficial targets for optimized strains, which ideally could be used for improved secretion of other proteins. We focused on an approach in the yeast Saccharomyces cerevisiae that allows for attenuation of gene expression, using RNAi combined with high-throughput microfluidic single-cell screening for cells with improved protein secretion. Using direct experimental validation or enrichment analysis-assisted characterization of systematically introduced RNAi perturbations, we could identify targets that improve protein secretion. We found that genes with functions in cellular metabolism (YDC1, AAD4, ADE8, and SDH1), protein modification and degradation (VPS73, KTR2, CNL1, and SSA1), and cell cycle (CDC39), can all impact recombinant protein production when expressed at differentially down regulated levels. By establishing a workflow that incorporates Cas9-mediated recombineering, we demonstrated how we could tune the expression of the identified gene targets for further improved protein production for specific proteins. Our findings offer a high throughput and semirational platform design, which will improve not only the production of a desired protein but even more importantly, shed additional light on connections between protein production and other cellular processes.

  • 48.
    Weibull, Emilie
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Bai, Yunpeng
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Jönsson, Håkan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Andersson Svahn, Helen
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Interfacing picoliter droplet microfluidics with addressable μl-compartments using FACS2013Ingår i: 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013, 2013, Vol. 3, s. 1632-1634Konferensbidrag (Refereegranskat)
    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.

1 - 48 av 48
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