| 1. |
- Rhedin, S. A., et al.
(författare)
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Protocol Introducing a New Algorithm for Classification of Etiology in Studies on Pediatric Pneumonia: Protocol for the Trial of Respiratory Infections in Children for Enhanced Diagnostics Study
- 2019
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Ingår i: Journal of Medical Internet Research. - : JMIR Publications Inc.. - 1438-8871. ; 21:4
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Tidskriftsartikel (refereegranskat)abstract
- Background: There is a need to better distinguish viral infections from antibiotic-requiring bacterial infections in children presenting with clinical community-acquired pneumonia (CAP) to assist health care workers in decision making and to improve the rational use of antibiotics. Objective: The overall aim of the Trial of Respiratory infections in children for ENhanced Diagnostics (TREND) study is to improve the differential diagnosis of bacterial and viral etiologies in children aged below 5 years with clinical CAP, by evaluating myxovirus resistance protein A (MxA) as a biomarker for viral CAP and by evaluating an existing (multianalyte point-of-care antigen detection test system [mariPOC respi] ArcDia International Oy Ltd.) and a potential future point-of-care test for respiratory pathogens. Methods: Children aged 1 to 59 months with clinical CAP as well as healthy, hospital-based, asymptomatic controls will be included at a pediatric emergency hospital in Stockholm, Sweden. Blood (analyzed for MxA and C-reactive protein) and nasopharyngeal samples (analyzed with real-time polymerase chain reaction as the gold standard and antigen-based mariPOC respi test as well as saved for future analyses of a novel recombinase polymerase amplification-based point-of-care test for respiratory pathogens) will be collected. A newly developed algorithm for the classification of CAP etiology will be used as the reference standard. Results: A pilot study was performed from June to August 2017. The enrollment of study subjects started in November 2017. Results are expected by the end of 2019.Conclusions: The findings from the TREND study can be an important step to improve the management of children with clinical. © 2019 Journal of Medical Internet Research. All rights reserved.
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| 2. |
- Afrasiabi, Roodabeh, et al.
(författare)
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Integration of a droplet-based microfluidic system and silicon nanoribbon FET sensor
- 2016
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Ingår i: Micromachines. - : MDPI AG. - 2072-666X. ; 7:8
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
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| 4. |
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| 5. |
- Andersson Svahn, Helene, et al.
(författare)
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Single cells or large populations?
- 2007
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 7:5, s. 544-546
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Tidskriftsartikel (refereegranskat)
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| 6. |
- Antypas, H., et al.
(författare)
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A universal platform for selection and high-resolution phenotypic screening of bacterial mutants using the nanowell slide
- 2018
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Ingår i: Lab on a Chip. - : ROYAL SOC CHEMISTRY. - 1473-0197 .- 1473-0189. ; 18:12, s. 1767-1777
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Tidskriftsartikel (refereegranskat)abstract
- The Petri dish and microtiter plate are the golden standard for selection and screening of bacteria in microbiological research. To improve on the limited resolution and throughput of these methods, we developed a universal, user-friendly platform for selection and high-resolution phenotypic screening based on the nanowell slide. This miniaturized platform has an optimal ratio between throughput and assay complexity, holding 672 nanowells of 500 nl each. As monoclonality is essential in bacterial genetics, we used FACS to inoculate each nanowell with a single bacterium in 15 min. We further extended the protocol to select and sort only bacteria of interest from a mixed culture. We demonstrated this by isolating single transposon mutants generated by a custom-made transposon with dual selection for GFP fluorescence and kanamycin resistance. Optical compatibility of the nanowell slide enabled phenotypic screening of sorted mutants by spectrophotometric recording during incubation. By processing the absorbance data with our custom algorithm, a phenotypic screen for growth-associated mutations was performed. Alternatively, by processing fluorescence data, we detected metabolism-associated mutations, exemplified by a screen for -galactosidase activity. Besides spectrophotometry, optical compatibility enabled us to perform microscopic analysis directly in the nanowells to screen for mutants with altered morphologies. Despite the miniaturized format, easy transition from nano- to macroscale cultures allowed retrieval of bacterial mutants for downstream genetic analysis, demonstrated here by a cloning-free single-primer PCR protocol. Taken together, our FACS-linked nanowell slide replaces manual selection of mutants on agar plates, and enables combined selection and phenotypic screening in a one-step process. The versatility of the nanowell slide, and the modular workflow built on mainstream technologies, makes our universal platform widely applicable in microbiological research.
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| 7. |
- Bai, Yunpeng, et al.
(författare)
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Interfacing picoliter droplet microfluidics with addressable microliter compartments using fluorescence activated cell sorting
- 2014
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Ingår i: Sensors and actuators. B, Chemical. - : Elsevier BV. - 0925-4005 .- 1873-3077. ; 194, s. 249-254
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Barbe, Laurent, et al.
(författare)
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Toward a confocal subcellular atlas of the human proteome
- 2008
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Ingår i: Molecular and cellular proteomics. - 1535-9476 .- 1535-9484. ; 7:3, s. 499-508
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Tidskriftsartikel (refereegranskat)abstract
- Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.
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| 9. |
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| 10. |
- Björk, Sara, 1990-
(författare)
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Droplet microfluidics for screening and sorting of microbial cell factories
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cell factories are cells that have been engineered to produce a compound of interest, ranging from biopharmaceuticals to biofuels. With advances in metabolic engineering, the number of cell factory variants to evaluate has increased dramatically, necessitating screening methods with increased throughput. Microfluidic droplets, which can be generated, manipulated and interrogated at very high throughput, are isolated reaction vessels at the single cell scale. Compartmentalization maintains the genotype-phenotype link, making droplet microfluidics suitable for screening of extracellular traits such as secreted products and for screening of microcolonies originating from single cells. In Paper I, we investigated the impact of droplet microfluidic incubation formats on cell culture conditions and found that syringe and semi open incubation resulted in different metabolic profiles. Controlling culture conditions is key to cell factory screening, as product formation is influenced by the state of the cell. In Paper II and III, we used droplet microfluidics to perform screening campaigns of interference based cell factory variant libraries. In Paper II, two S. cerevisiae RNAi libraries were screened based on amylase secretion, and from the sorted fraction genes linked to improved protein secretion could be identified. In paper III, we screened a Synecosystis sp. CRISPRi library based on lactate secretion. The library was sorted at different time points after induction, followed by sequencing to reveal genes enriched by droplet sorting. In Paper IV, we developed a droplet microcolony-based assay for screening intracellular triacylglycerol (TAG) in S. cerevisiae, and showed improved strain separation compared to flow cytometry in a hypothetical sorting scenario. By screening microcolonies compartmentalized in droplets, we combine the throughput of single cell screening methods with the reduced impact of cell-to-cell noise in cell ensemble analysis.
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| 11. |
- Björk, Sara M., et al.
(författare)
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Metabolite profiling of microfluidic cell culture conditions for droplet based screening
- 2015
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Ingår i: Biomicrofluidics. - : AIP Publishing. - 1932-1058. ; 9:4
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 12. |
- Björk, S. M., et al.
(författare)
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Tuning microfluidic cell culture conditions for droplet based screening by metabolite profiling
- 2015
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Ingår i: MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - : Chemical and Biological Microsystems Society. - 9780979806483 ; , s. 1377-1379
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Konferensbidrag (refereegranskat)abstract
- 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.
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| 13. |
- Chinnasamy, Thiruppathiraja, et al.
(författare)
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A lateral flow paper microarray for rapid allergy point of care diagnostics
- 2014
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Ingår i: The Analyst. - : Royal Society of Chemistry. - 0003-2654 .- 1364-5528. ; 139:10, s. 2348-2354
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Tidskriftsartikel (refereegranskat)abstract
- There is a growing need for multiplexed specific IgE tests that can accurately evaluate patient sensitization profiles. However, currently available commercial tests are either single/low-plexed or require sophisticated instrumentation at considerable cost per assay. Here, we present a novel convenient lateral flow microarray-based device that employs a novel dual labelled gold nanoparticle-strategy for rapid and sensitive detection of a panel of 15 specific IgE responses in 35 clinical serum samples. Each gold nanoparticle was conjugated to an optimized ratio of HRP and anti-IgE, allowing significant enzymatic amplification to improve the sensitivity of the assay as compared to commercially available detection reagents. The mean inter-assay variability of the developed LFM assay was 12% CV, and analysis of a cohort of clinical samples (n = 35) revealed good general agreement with ImmunoCAP, yet with a varying performance among allergens (AUC = [0.54-0.88], threshold 1 kU). Due to the rapid and simple procedure, inexpensive materials and read-out by means of a consumer flatbed scanner, the presented assay may provide an interesting low-cost alternative to existing multiplexed methods when thresholds > 1 kU are acceptable.
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| 14. |
- Chinnasamy, Thiruppathiraja, et al.
(författare)
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Point-of-Care Vertical Flow Allergen Microarray Assay : Proof of Concept
- 2014
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 60:9, s. 1209-1216
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Sophisticated equipment, lengthy protocols, and skilled operators are required to perform protein microarray-based affinity assays. Consequently, novel tools are needed to bring biomarkers and biomarker panels into clinical use in different settings. Here, we describe a novel paper-based vertical flow microarray (VFM) system with a multiplexing capacity of at least 1480 microspot binding sites, colorimetric readout, high sensitivity, and assay time of < 10 min before imaging and data analysis. METHOD: Affinity binders were deposited on nitrocellulose membranes by conventional microarray printing. Buffers and reagents were applied vertically by use of a flow controlled syringe pump. As a clinical model system, we analyzed 31 precharacterized human serum samples using the array system with 10 allergen components to detect specific IgE reactivities. We detected bound analytes using gold nanoparticle conjugates with assay time of <= 10 min. Microarray images were captured by a consumer-grade flatbed scanner. RESULTS: A sensitivity of 1 ng/mL was demonstrated with the VFM assay with colorimetric readout. The reproducibility (CV) of the system was < 14%. The observed concordance with a clinical assay, Immuno-CAP, was R-2 = 0.89 (n = 31). CONCLUSIONS: In this proof-of-concept study, we demonstrated that the VFM assay, which combines features from protein microarrays and paper-based colorimetric systems, could offer an interesting alternative for future highly multiplexed affinity point-of-care testing.
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| 15. |
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| 16. |
- Dias, Jorge, et al.
(författare)
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Rapid nanoprobe signal enhancement by in situ gold nanoparticle synthesis
- 2018
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Ingår i: Journal of Visualized Experiments. - : Journal of Visualized Experiments. - 1940-087X. ; 2018:133
-
Tidskriftsartikel (refereegranskat)abstract
- The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good's buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera.
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| 17. |
- Dias, Jorge T., et al.
(författare)
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Minimizing antibody cross-reactivity in multiplex detection of biomarkers in paper-based point-of-care assays
- 2016
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Ingår i: Nanoscale. - : Royal Society of Chemistry. - 2040-3364 .- 2040-3372. ; 8:15, s. 8195-8201
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Tidskriftsartikel (refereegranskat)abstract
- Highly multiplexed immunoassays could allow convenient screening of hundreds or thousands of protein biomarkers simultaneously in a clinical sample such as serum or plasma, potentially allowing improved diagnostic accuracy and clinical management of many conditions such as autoimmune disorders, infections, and several cancers. Currently, antibody microarray-based tests are limited in part due to cross reactivity from detection antibody reagents. Here we present a strategy that reduces the cross-reactivity between nanoparticle-bound reporter antibodies through the application of ultrasound energy. By this concept, it was possible to achieve a sensitivity 10(3)-fold (5 pg mL(-1)) lower than when no ultrasound was applied (50 ng mL(-1)) for the simultaneous detection of three different antigens. The detection limits and variability achieved with this technique rival those obtained with other types of multiplex sandwich assays.
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| 18. |
- Dias, Jorge T, et al.
(författare)
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Rapid signal enhancement method for nanoprobe-based biosensing
- 2017
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 7
-
Tidskriftsartikel (refereegranskat)abstract
- The introduction of nanomaterials as detection reagents has enabled improved sensitivity and facilitated detection in a variety of bioanalytical assays. However, high nanoprobe densities are typically needed for colorimetric detection and to circumvent this limitation several enhancement protocols have been reported. Nevertheless, there is currently a lack of universal, enzyme-free and versatile methods that can be readily applied to existing as well as new biosensing strategies. The novel method presented here is shown to enhance the signal of gold nanoparticles enabling visual detection of a spot containing < 10 nanoparticles. Detection of Protein G on paper arrays was improved by a 100-fold amplification factor in under five minutes of assay time, using IgG-labelled gold, silver, silica and iron oxide nanoprobes. Furthermore, we show that the presented protocol can be applied to a commercial allergen microarray assay, ImmunoCAP ISAC sIgE 112, attaining a good agreement with fluorescent detection when analysing human clinical samples.
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| 19. |
- Dias, Jorge T., et al.
(författare)
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Rapid signal enhancement method for nanoprobe-based biosensing (vol 7, 2017)
- 2018
-
Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 8:1
-
Tidskriftsartikel (refereegranskat)abstract
- In the Methods section of this Article references 18 to 22 are incorrectly cited. The correct references were omitted from the reference list and appear below as references 1-5. References 18 to 22 are correctly cited in Introduction and Results and Discussion sections. "AuNPs of 10 nm in diameter were prepared following the protocol described by Bastus et al.18." should read: "AuNPs of 10 nm in diameter were prepared following the protocol described by Bastus et al.1." "AgNPs of 90 nm in diameter were prepared following the protocol described by Rivero et al.19." should read: "AgNPs of 90 nm in diameter were prepared following the protocol described by Rivero et al.2" "The size was determined by UV-Vis spectroscopy according to the AgNPs size theory demonstrated by Malynych20." should read: "The size was determined by UV-Vis spectroscopy according to the AgNPs size theory demonstrated by Malynych3." "The coupling of antibody to the NPs was prepared following a modified version of a protocol previously reported by Puertas et al.21." should read: "The coupling of antibody to the NPs was prepared following a modified version of a protocol previously reported by Puertas et al.4." "Microarrays were prepared as previously reported by our group22." should read: "Microarrays were prepared as previously reported by our group5.
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| 20. |
- Douagi, Anna Sjöström, et al.
(författare)
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Young researchers to tackle future Grand Challenges
- 2012
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 12:4, s. 680-683
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 21. |
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| 22. |
- Eissler, Nina, et al.
(författare)
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Regulation of myeloid cells by activated T cells determines the efficacy of PD-1 blockade
- 2016
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Ingår i: Oncoimmunology. - : Taylor & Francis. - 2162-4011 .- 2162-402X. ; 5:12
-
Tidskriftsartikel (refereegranskat)abstract
- Removal of immuno-suppression has been reported to enhance antitumor immunity primed by checkpoint inhibitors. Although PD-1 blockade failed to control tumor growth in a transgenic murine neuroblastoma model, concurrent inhibition of colony stimulating factor 1 receptor (CSF-1R) by BLZ945 reprogrammed suppressive myeloid cells and significantly enhanced therapeutic effects. Microarray analysis of tumor tissues identified a significant increase of T-cell infiltration guided by myeloid cell-derived chemokines CXCL9, 10, and 11. Blocking the responsible chemokine receptor CXCR3 hampered T-cell infiltration and reduced antitumor efficacy of the combination therapy. Multivariate analysis of 59 immune-cell parameters in tumors and spleens detected the correlation between PD-L1-expressing myeloid cells and tumor burden. In vitro, anti-PD-1 antibody Nivolumab in combination with BLZ945 increased the activation of primary human T and NK cells. Importantly, we revealed a previously uncharacterized pathway, in which T cells secreted M-CSF upon PD-1 blockade, leading to enhanced suppressive capacity of monocytes by upregulation of PD-L1 and purinergic enzymes. In multiple datasets of neuroblastoma patients, gene expression of CD73 correlated strongly with myeloid cell markers CD163 and CSF-1R in neuroblastoma tumors, and associated with worse survival in high-risk patients. Altogether, our data reveal the dual role of activated T cells on myeloid cell functions and provide a rationale for the combination therapy of anti-PD-1 antibody with CSF-1R inhibitor.
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| 23. |
- Friedman, Mikaela, et al.
(författare)
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Engineering and characterization of a bispecific HER2 x EGFR-binding affibody molecule
- 2009
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Ingår i: Biotechnology and applied biochemistry. - 0885-4513 .- 1470-8744. ; 54, s. 121-131
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 24. |
- Frisk, Thomas, et al.
(författare)
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A microfluidic device for parallel 3-D cell cultures in asymmetric environments
- 2007
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 28:24, s. 4705-4712
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 25. |
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| 26. |
- Gantelius, Jesper, et al.
(författare)
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A Lateral Flow Protein Microarray for Rapid and Sensitive Antibody Assays
- 2011
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 12:11, s. 7748-7759
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 27. |
- Gantelius, Jesper, et al.
(författare)
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A lateral flow protein microarray for rapid determination of contagious bovine pleuropneumonia status in bovine serum
- 2010
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 82:1, s. 11-18
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 28. |
- Gantelius, Jesper, et al.
(författare)
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A ten-minute high density lateral flow protein microarray assay
- 2011
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Ingår i: 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011. - 9781618395955 ; , s. 1176-1178
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Konferensbidrag (refereegranskat)abstract
- 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.
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| 29. |
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| 30. |
- Gantelius, Jesper, et al.
(författare)
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Improved sensitivity on an allergen lateral flow microarray by means of dendritic amplification
- 2009
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Ingår i: Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - : Chemical and Biological Microsystems Society. - 9780979806421 ; , s. 1136-1137
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Konferensbidrag (refereegranskat)abstract
- 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.
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| 31. |
- Gantelius, Jesper, et al.
(författare)
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Magnetic bead-based detection of autoimmune responses using protein microarrays.
- 2009
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Ingår i: New biotechnology. - : Elsevier BV. - 1871-6784. ; 26, s. 269-276
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 32. |
- Gantelius, Jesper, 1980-
(författare)
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Novel diagnostic microarray assay formats towards comprehensive on-site analysis
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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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| 33. |
- Gaudenzi, Giulia, et al.
(författare)
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Point-of-Care Approaches for Meningitis Diagnosis in a Low-Resource Setting (Southwestern Uganda) : Observational Cohort Study Protocol of the "PI-POC" Trial
- 2020
-
Ingår i: Journal of Medical Internet Research. - : JMIR Publications Inc.. - 1438-8871. ; 22:11
-
Tidskriftsartikel (refereegranskat)abstract
- Background: A timely differential diagnostic is essential to identify the etiology of central nervous system (CNS) infections in children, in order to facilitate targeted treatment, manage patients, and improve clinical outcome. Objective: The Pediatric Infection-Point-of-Care (PI-POC) trial is investigating novel methods to improve and strengthen the differential diagnostics of suspected childhood CNS infections in low-income health systems such as those in Southwestern Uganda. This will be achieved by evaluating (1) a novel DNA-based diagnostic assay for CNS infections, (2) a commercially available multiplex PCR-based meningitis/encephalitis (ME) panel for clinical use in a facility-limited laboratory setting, (3) proteomics profiling of blood from children with severe CNS infection as compared to outpatient controls with fever yet not severely ill, and (4) Myxovirus resistance protein A (MxA) as a biomarker in blood for viral CNS infection. Further changes in the etiology of childhood CNS infections after the introduction of the pneumococcal conjugate vaccine against Streptococcus pneumoniae will be investigated. In addition, the carriage and invasive rate of Neisseria meningitidis will be recorded and serotyped, and the expression of its major virulence factor (polysaccharide capsule) will be investigated. Methods: The PI-POC trial is a prospective observational study of children including newborns up to 12 years of age with clinical features of CNS infection, and age-/sex-matched outpatient controls with fever yet not severely ill. Participants are recruited at 2 Pediatric clinics in Mbarara, Uganda. Cerebrospinal fluid (for cases only), blood, and nasopharyngeal (NP) swabs (for both cases and controls) sampled at both clinics are analyzed at the Epicentre Research Laboratory through gold-standard methods for CNS infection diagnosis (microscopy, biochemistry, and culture) and a commercially available ME panel for multiplex PCR analyses of the cerebrospinal fluid. An additional blood sample from cases is collected on day 3 after admission. After initial clinical analyses in Mbarara, samples will be transported to Stockholm, Sweden for (1) validation analyses of a novel nucleic acid-based POC test, (2) biomarker research, and (3) serotyping and molecular characterization of S. pneumoniae and N. meningitidis. Results: A pilot study was performed from January to April 2019. The PI-POC trial enrollment of patients begun in April 2019 and will continue until September 2020, to include up to 300 cases and controls. Preliminary results from the PI-POC study are expected by the end of 2020. Conclusions: The findings from the PI-POC study can potentially facilitate rapid etiological diagnosis of CNS infections in low-resource settings and allow for novel methods for determination of the severity of CNS infection in such environment.
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| 34. |
- Guldevall, Karolin, et al.
(författare)
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Imaging Immune Surveillance of Individual Natural Killer Cells Confined in Microwell Arrays
- 2010
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 5:11, s. e15453-
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 35. |
- Hammond, Maria, et al.
(författare)
-
Picodroplet partitioned whole genome amplification of low biomass samples preserves genomic diversity for metagenomic analysis
- 2016
-
Ingår i: Microbiome. - : BioMed Central. - 2049-2618. ; 4
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 36. |
- Huang, Mingtao, 1984, et al.
(författare)
-
Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast
- 2015
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 112:34, s. E4689-E4696
-
Tidskriftsartikel (refereegranskat)abstract
- 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 high-throughput 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 a-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome 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.
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| 37. |
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| 38. |
- Joensson, Haakan, et al.
(författare)
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Concurrent multi-sample analysis of low expressed biomarkers on single human cells by enzymatically amplified immunodetection in droplets
- 2008
-
Ingår i: 12th International Conference on Miniaturized Systems for Chemistry and Life Sciences - The Proceedings of MicroTAS 2008 Conference. - : Chemical and Biological Microsystems Society. ; , s. 1287-1289
-
Konferensbidrag (refereegranskat)abstract
- 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.
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| 39. |
- Jönsson, Håkan, et al.
(författare)
-
A homogeneous assay for biomolecule interaction analysis in droplets by flourescence polarization
- 2010
-
Ingår i: 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010. - 9781618390622 ; , s. 1802-1804
-
Konferensbidrag (refereegranskat)abstract
- 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.
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| 40. |
- Jönsson, Håkan, 1979-, et al.
(författare)
-
A Homogeneous Assay for Protein Analysis in Droplets by Fluorescence Polarization
- 2012
-
Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 33:3, s. 436-439
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 41. |
- Jönsson, Håkan, et al.
(författare)
-
Detection and Analysis of Low-Abundance Cell-Surface Biomarkers Using Enzymatic Amplification in Microfluidic Droplets
- 2009
-
Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 48:14, s. 2518-2521
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 42. |
- Jönsson, Håkan, et al.
(författare)
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Deterministic lateral displacement device for droplet separation by size - Towards rapid clonal selection based on droplet shrinking
- 2010
-
Ingår i: 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010. - 9781618390622 ; , s. 1355-1357
-
Konferensbidrag (refereegranskat)abstract
- 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].
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| 43. |
- Jönsson, Håkan, 1979-
(författare)
-
Droplet microfluidics for high throughput biological analysis
- 2011
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 44. |
- Jönsson, Håkan, 1979-, et al.
(författare)
-
Droplet size based separation by deterministic lateral displacement : separating droplets by cell-induced shrinking
- 2011
-
Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 11:7, s. 1305-1310
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 45. |
- Jönsson, Håkan, et al.
(författare)
-
Microfluidic droplet based enzyme variant screening : Towards improved enzymes for industrial applications
- 2011
-
Ingår i: 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011. - 9781618395955 ; , s. 179-181
-
Konferensbidrag (refereegranskat)abstract
- 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.
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| 46. |
|
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| 47. |
- Jönsson, Håkan N., et al.
(författare)
-
Droplet microfluidics-A tool for single-cell analysis
- 2012
-
Ingår i: Angewandte Chemie International Edition. - : Wiley-VCH Verlagsgesellschaft. - 1433-7851 .- 1521-3773. ; 51:49, s. 12176-12192
-
Forskningsöversikt (refereegranskat)abstract
- 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.
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| 48. |
- Jönsson, Håkan, 1979-, et al.
(författare)
-
Tröpfchen-Mikrofluidik für die Einzelzellanalyse
- 2012
-
Ingår i: Angewandte Chemie. - : Wiley Online Library. - 0044-8249 .- 1521-3757. ; 124:49, s. 12342-12359
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 49. |
- Khati, Vamakshi, et al.
(författare)
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3D Bioprinting of Multi-Material Decellularized Liver Matrix Hydrogel at Physiological Temperatures
- 2022
-
Ingår i: Biosensors. - : MDPI AG. - 2079-6374. ; 12:7
-
Tidskriftsartikel (refereegranskat)abstract
- Bioprinting is an acclaimed technique that allows the scaling of 3D architectures in an organized pattern but suffers from a scarcity of appropriate bioinks. Decellularized extracellular matrix (dECM) from xenogeneic species has garnered support as a biomaterial to promote tissue-specific regeneration and repair. The prospect of developing dECM-based 3D artificial tissue is impeded by its inherent low mechanical properties. In recent years, 3D bioprinting of dECM-based bioinks modified with additional scaffolds has advanced the development of load-bearing constructs. However, previous attempts using dECM were limited to low-temperature bioprinting, which is not favorable for a longer print duration with cells. Here, we report the development of a multi-material decellularized liver matrix (dLM) bioink reinforced with gelatin and polyethylene glycol to improve rheology, extrudability, and mechanical stability. This shear-thinning bioink facilitated extrusion-based bioprinting at 37 degrees C with HepG2 cells into a 3D grid structure with a further enhancement for long-term applications by enzymatic crosslinking with mushroom tyrosinase. The heavily crosslinked structure showed a 16-fold increase in viscosity (2.73 Pa s(-1)) and a 32-fold increase in storage modulus from the non-crosslinked dLM while retaining high cell viability (85-93%) and liver-specific functions. Our results show that the cytocompatible crosslinking of dLM bioink at physiological temperatures has promising applications for extended 3D-printing procedures.
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| 50. |
- Khati, Vamakshi, et al.
(författare)
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A tunable decellularized liver-based hybrid bioink
- 2021
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Ingår i: MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - : Chemical and Biological Microsystems Society. ; , s. 281-282
-
Konferensbidrag (refereegranskat)abstract
- Decellularized extracellular matrix is a tissue-specific biomaterial that recapitulates the complexity of the inherent tissue environment to elicit cellular response. Here, a multi-material decellularized liver (dLM)-based bioink with gelatin is developed and polyethylene glycol crosslinking is used to enhance the viscoelasticity of the dLM. We evaluated the necessity of a post-printing secondary cross-linker mushroom tyrosinase to improve robustness and long term stability. We further demonstrate it's biocompatibility using liver specific gene analysis of HepG2 cells.
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