| 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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| 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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