| 1. |
- 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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| 2. |
- Armbrecht, L., et al.
(författare)
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Self-assembled magnetic bead chains for sensitivity enhancement of microfluidic electrochemical biosensor platforms
- 2015
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 15:22, s. 4314-4321
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Tidskriftsartikel (refereegranskat)abstract
- In this paper, we present a novel approach to enhance the sensitivity of microfluidic biosensor platforms with self-assembled magnetic bead chains. An adjustable, more than 5-fold sensitivity enhancement is achieved by introducing a magnetic field gradient along a microfluidic channel by means of a soft-magnetic lattice with a 350 mu m spacing. The alternating magnetic field induces the self-assembly of the magnetic beads in chains or clusters and thus improves the perfusion and active contact between the analyte and the beads. The soft-magnetic lattices can be applied independent of the channel geometry or chip material to any microfluidic biosensing platform. At the same time, the bead-based approach achieves chip reusability and shortened measurement times. The bead chain properties and the maximum flow velocity for bead retention were validated by optical microscopy in a glass capillary. The magnetic actuation system was successfully validated with a biotin-streptavidin model assay on a low-cost electrochemical microfluidic chip, fabricated by dry-film photoresist technology (DFR). Labelling with glucose oxidase (GOx) permits rapid electrochemical detection of enzymatically produced H2O2.
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| 3. |
- Banerjee, Indradumna, et al.
(författare)
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Microfluidic centrifugation assisted precipitation based DNA quantification
- 2019
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry. - 1473-0197 .- 1473-0189. ; 19:9, s. 1657-1664
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Tidskriftsartikel (refereegranskat)abstract
- Nucleic acid amplification methods are increasingly being used to detect trace quantities of DNA in samples for various diagnostic applications. However, quantifying the amount of DNA from such methods often requires time consuming purification, washing or labeling steps. Here, we report a novel microfluidic centrifugation assisted precipitation (mu CAP) method for single-step DNA quantification. The method is based on formation of a visible precipitate, which can be quantified, when an intercalating dye (GelRed) is added to the DNA sample and centrifuged for a few seconds. We describe the mechanism leading to the precipitation phenomenon. We utilize centrifugal microfluidics to precisely control the formation of the visible and quantifiable mass. Using a standard CMOS sensor for imaging, we report a detection limit of 45 ng mu l(-1). Furthermore, using an integrated lab-on-DVD platform we recently developed, the detection limit is lowered to 10 ng mu l(-1), which is comparable to those of current commercially available instruments for DNA quantification. As a proof of principle, we demonstrate the quantification of LAMP products for a HIV-1B type genome containing plasmid on the lab-on-DVD platform. The simple DNA quantification system could facilitate advanced point of care molecular diagnostics.
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| 4. |
- Bergström, Gunnar, et al.
(författare)
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Stem cell derived in vivo-like human cardiac bodies in a microfluidic device for toxicity testing by beating frequency imaging
- 2015
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry. - 1473-0197 .- 1473-0189. ; 15:15, s. 3242-3249
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Tidskriftsartikel (refereegranskat)abstract
- Beating in vivo-like human cardiac bodies (CBs) were used in a microfluidic device for testing cardiotoxicity. The CBs, cardiomyocyte cell clusters derived from induced pluripotent stem cells, exhibited typical structural and functional properties of the native human myocardium. The CBs were captured in niches along a perfusion channel in the device. Video imaging was utilized for automatic monitoring of the beating frequency of each individual CB. The device allowed assessment of cardiotoxic effects on the 3D clustered cardiomyocytes from the drug substances doxorubicin, verapamil and quinidine. Beating frequency data recorded over a period of 6 hours are presented and compared to literature data. The results indicate that this microfluidic setup with imaging of CB characteristics provides a new opportunity for label-free, non-invasive investigation of toxic effects in a 3D microenvironment.
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| 5. |
- Bryl-Górecka, Paulina, et al.
(författare)
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Effect of exercise on the plasma vesicular proteome : A methodological study comparing acoustic trapping and centrifugation
- 2018
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 18:20, s. 3101-3111
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular vesicles (EVs) are a heterogeneous group of actively released vesicles originating from a wide range of cell types. Characterization of these EVs and their proteomes in the human plasma provides a novel approach in clinical diagnostics, as they reflect physiological and pathological states. However, EV isolation is technically challenging with the current methods having several disadvantages, requiring large sample volumes, and resulting in loss of sample and EV integrity. Here, we use an alternative, non-contact method based on a microscale acoustic standing wave technology. Improved coupling of the acoustic resonator increased the EV recovery from 30% in earlier reports to 80%, also displaying long term stability between experiment days. We report a pilot study, with 20 subjects who underwent physical exercise. Plasma samples were obtained before and 1 h after the workout. Acoustic trapping was compared to a standard high-speed centrifugation protocol, and the method was validated by flow cytometry (FCM). To monitor the device stability, the pooled frozen plasma from volunteers was used as an internal control. A key finding from the FCM analysis was a decrease in CD62E+ (E-selectin) EVs 1 h after exercise that was consistent for both methods. Furthermore, we report the first data that analyse differential EV protein expression before and after physical exercise. Olink-based proteomic analysis showed 54 significantly changed proteins in the EV fraction in response to physical exercise, whereas the EV-free plasma proteome only displayed four differentially regulated proteins, thus underlining an important role of these vesicles in cellular communication, and their potential as plasma derived biomarkers. We conclude that acoustic trapping offers a fast and efficient method comparable with high-speed centrifugation protocols. Further, it has the advantage of using smaller sample volumes (12.5 μL) and rapid contact-free separation with higher yield, and can thus pave the way for future clinical EV-based diagnostics.
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| 6. |
- Christakou, Athanasia, et al.
(författare)
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Ultrasonic three-dimensional on-chip cell culture for dynamic studies of tumor immune surveillance by natural killer cells
- 2015
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry. - 1473-0197 .- 1473-0189. ; 15:15, s. 3222-31
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate a simple method for three-dimensional (3D) cell culture controlled by ultrasonic standing waves in a multi-well microplate. The method gently arranges cells in a suspension into a single aggregate in each well of the microplate and, by this, nucleates 3D tissue-like cell growth for culture times between two and seven days. The microplate device is compatible with both high-resolution optical microscopy and maintenance in a standard cell incubator. The result is a scaffold- and coating-free method for 3D cell culture that can be used for controlling the cellular architecture, as well as the cellular and molecular composition of the microenvironment in and around the formed cell structures. We demonstrate the parallel production of one hundred synthetic 3D solid tumors comprising up to thousands of human hepatocellular carcinoma (HCC) HepG2 cells, we characterize the tumor structure by high-resolution optical microscopy, and we monitor the functional behavior of natural killer (NK) cells migrating, docking and interacting with the tumor model during culture. Our results show that the method can be used for determining the collective ability of a given number of NK cells to defeat a solid tumor having a certain size, shape and composition. The ultrasound-based method itself is generic and can meet any demand from applications where it is advantageous to monitor cell culture from production to analysis of 3D tissue or tumor models using microscopy in one single microplate device.
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| 7. |
- Cruz, Javier, 1990-, et al.
(författare)
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Inertial focusing with sub-micron resolution for separation of bacteria
- 2019
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Ingår i: Lab on a Chip. - : ROYAL SOC CHEMISTRY. - 1473-0197 .- 1473-0189. ; 19:7, s. 1257-1266
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Tidskriftsartikel (refereegranskat)abstract
- In this paper, we study inertial focusing in curved channels and demonstrate the alignment of particles with diameters between 0.5 and 2.0 m, a range of biological relevance since it comprises a multitude of bacteria and organelles of eukaryotic cells. The devices offer very sensitive control over the equilibrium positions and allow two modes of operation. In the first, particles having a large variation in size are focused and concentrated together. In the second, the distribution spreads in a range of sizes achieving separation with sub-micron resolution. These systems were validated with three bacteria species (Escherichia coli, Salmonella typhimurium and Klebsiella pneumoniae) showing good alignment while maintaining the viability in all cases. The experiments also revealed that the particles follow a helicoidal trajectory to reach the equilibrium positions, similar to the fluid streamlines simulated in COMSOL, implying that these positions occupy different heights in the cross section. When the equilibrium positions move to the inner wall as the flow rate increases, they are at a similar distance from the centre than in straight channels (approximate to 0.6R), but when the equilibrium positions move to the outer wall as the flow rate increases, they are closer to the centre and the particles pass close to the inner wall to elevate their position before reaching them. These observations were used along with COMSOL simulations to explain the mechanism behind the local force balance and the migration of particles, which we believe contributes to further understanding of the phenomenon. Hopefully, this will make designing more intuitive and reduce the high pressure demands, enabling manipulation of particles much smaller than a micrometer.
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| 8. |
- Evander, Mikael, et al.
(författare)
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Non-contact acoustic capture of microparticles from small plasma volumes.
- 2015
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0189 .- 1473-0197. ; 15:12, s. 2588-2596
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Tidskriftsartikel (refereegranskat)abstract
- Microparticles (MP) are small (100-1000 nm) membrane vesicles shed by cells as a response to activation, stress or apoptosis. Platelet-derived MP (PMP) has been shown to reflect the pathophysiological processes of a range of cardiovascular diseases and there is a potential clinical value in using PMPs as biomarkers, as well as a need to better understand the biology of these vesicles. The current method for isolating MP depends on differential centrifugation steps, which require relatively large sample volumes and have been shown to compromise the integrity and composition of the MP population. We present a novel method for rapid, non-contact capture of PMP in minute sample volumes based on a microscale acoustic standing wave technology. Capture of PMPs from plasma is shown by scanning electron microscopy and flow cytometry. Furthermore, the system is characterized with regards to plasma sample concentration and flow rate. Finally, the technique is compared to a standard differential centrifugation protocol using samples from both healthy controls and ST-elevation myocardial infarction (STEMI) patient samples. The acoustic system is shown to offer a quick and automated setup for extracting microparticles from small sample volumes with higher recovery than a standard differential centrifugation protocol.
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| 9. |
- Fatsis-Kavalopoulos, Nikos, et al.
(författare)
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Formation of precisely composed cancer cell clusters using a cell assembly generator (CAGE) for studying paracrine signaling at single-cell resolution
- 2019
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Ingår i: Lab on a Chip. - : ROYAL SOC CHEMISTRY. - 1473-0197 .- 1473-0189. ; 19:6, s. 1071-1081
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Tidskriftsartikel (refereegranskat)abstract
- The function and behaviour of any given cell in a healthy tissue, or in a tumor, is affected by interactions with its neighboring cells. It is therefore important to create methods that allow for reconstruction of tissue niches in vitro for studies of cell-cell signaling and associated cell behaviour. To this end we created the cell assembly generator (CAGE), a microfluidic device which enables the organization of different cell types into precise cell clusters in a flow chamber compatible with high-resolution microscopy. In proof-of-concept paracrine signalling experiments, 4-cell clusters consisting of one pancreatic -cell and three breast cancer cells were formed. It has previously been established that extracellular ATP induces calcium (Ca2+) release from the endoplasmic reticulum (ER) to the cytosol before it is cleared back into the ER via sarcoplasmic/ER Ca2+ ATPase (SERCA) pumps. Here, ATP release from the -cell was stimulated by depolarization, and dynamic changes in Ca2+ levels in the adjacent cancer cells measured using imaging of the calcium indicator Fluo-4. We established that changes in the concentration of cytosolic Ca2+ in the cancer cells were proportional to the distance from the ATP-releasing -cell. Additionally, we established that the relationship between distance and cytosolic calcium changes were dependent on Ca2+-release from the ER using 5-cell clusters composed of one -cell, two untreated cancer cells and two cancer cells pretreated with Thapsigargin (to deplete the ER of Ca2+). These experiments show that the CAGE can be used to create exact cell clusters, which affords precise control for reductionist studies of cell-cell signalling and permits the formation of heterogenous cell models of specific tissue niches.
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| 10. |
- Friedrich, R., et al.
(författare)
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A nano flow cytometer for single lipid vesicle analysis
- 2017
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Ingår i: Lab on a Chip - Miniaturisation for Chemistry and Biology. - : Royal Society of Chemistry (RSC). - 1473-0189 .- 1473-0197. ; 17:5, s. 830-841
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Tidskriftsartikel (refereegranskat)abstract
- We present a nanofluidic device for fluorescence-based detection and characterization of small lipid vesicles on a single particle basis. The device works like a nano flow cytometer where individual vesicles are visualized by fluorescence microscopy while passing through parallel nanochannels in a pressure-driven flow. An experiment requires less than 20 mu l sample volume to quantify both the vesicle content and the fluorescence signals emitted by individual vesicles. We show that the device can be used to accurately count the number of fluorescent synthetic lipid vesicles down to a vesicle concentration of 170 fM. We also show that the size-distribution of the vesicles can be resolved from their fluorescence intensity distribution after calibration. We demonstrate the applicability of the assay in two different examples. In the first, we use the nanofluidic device to determine the particle concentration in a sample containing cell-derived extracellular vesicles labelled with a lipophilic dye. In the second, we demonstrate that dual-color detection can be used to probe peptide binding to synthetic lipid vesicles; we identify a positive membrane-curvature sensing behavior of an arginine enriched version of the Antennapedia homeodomain peptide penetratin. Altogether, these results illustrate the potential of this nanofluidic-based methodology for characterization and quantification of small biological vesicles and their interactors without ensemble averaging. The device is therefore likely to find use as a quantitative analytical tool in a variety of fields ranging from diagnostics to fundamental biology research. Moreover, our results have potential to facilitate further development of automated lab-on-a-chip devices for vesicle analysis.
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