Increasing the fungicidal action of Amphotericin B by inhibiting the Nitric Oxide-Dependent tolerance pathway

Amphotericin B (AmB) induces oxidative and nitrosative stresses, characterized by production of reactive oxygen and nitrogen species, in fungi. Yet, how these toxic species contribute to AmB-induced fungal cell death is unclear. We investigated the role of superoxide and nitric oxide radicals in AmB's fungicidal activity in Saccharomyces cerevisiae, using a digital microfluidic platform, which enabled monitoring individual cells at a spatiotemporal resolution, and plating assays. The nitric oxide synthase inhibitor L-NAME was used to interfere with nitric oxide radical production. L-NAME increased and accelerated AmB-induced accumulation of superoxide radicals, membrane permeabilization, and loss of proliferative capacity in S. cerevisiae. In contrast, the nitric oxide donor S-nitrosoglutathione inhibited AmB's action. Hence, superoxide radicals were important for AmB's fungicidal action, whereas nitric oxide radicals mediated tolerance towards AmB. Finally, also the human pathogens Candida albicans and Candida glabrata were more susceptible to AmB in the presence of L-NAME, pointing to the potential of AmB-L-NAME combination therapy to treat fungal infections.Kim Vriens acknowledges the receipt of a predoctoral grant from the Flanders Innovation & Entrepeneurship Agency (IWT-SB 111016); Karin Thevissen acknowledges the receipt of a mandate of Industrial Research Fund (KU Leuven). In addition, the research leading to these results has received funding from the Research Foundation - Flanders (FWO G086114N and G080016N) and the KU Leuven (OT 13/ 058 and IDO 10/012, IOF KP/12/009 Atheromix, IOF KP/ 12/002 Nanodiag). This work was partially developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). Belém Sampaio-Marques is supported by the fellowship SFRH/BPD/90533/2012 funded by Fundação para a Ciência e Tecnologia (FCT, Portugal).info:eu-repo/semantics/publishedVersio

Optical bead manipulation on a digital microfluidic chip for screening biomolecular interactions

status: publishe

Mr. Fabian Rempfer

https://thekeep.eiu.edu/commencement_fall2017/1039/thumbnail.jp

Digital Microfluidic chip technology for water permeability measurements on individual protoplasts

Osmotic potential studies are conventionally performed using micropipettes[1] or micromanipulators[2] guided through a microscope where cells are sequentially analyzed one at the time. The latest technological advancements offer promising alternatives for this type of assays. Electrowetting-on-dielectric (EWOD) based digital microfluidic (DMF) chips are platforms for the manipulation of individual microliter-sized droplets on a planar array of electrodes. They allow for single cell analysis (SCA) enabling a substantial increase of the number of measured cells per analysis. The application of EWOD-based DMF chip[3] is presented to perform osmotic potential studies on an array of single cells at once, using Arabidopsis Thaliana protoplasts as a model system[4]. Protoplasts were immobilized on-chip using magnetic particles[5,6] and exposed to different osmotic conditions in an automated way. The changes in protoplast volume was monitored simultaneously with the change of osmolarity. Osmotic water permeability coefficient[7] values of single protoplasts were extrapolated from the measured volumetric changes over time. This application demonstrates that the DMF platforms has great potential not only for osmotic studies but also for other types of SCA.status: publishe

A digital microfluidic platform for capture and selective retrieval of single bacteria

With the advent of powerful and automated fluorescence imaging techniques, in depth characterization of bacteria has become feasible. Although phenotypic screening is possible with the existing techniques, retrieval of interesting bacteria from a larger population for their subsequent analysis is still challenging. Moreover, the handling of such small cells suffers from low throughput and high manual work. Hence, novel technologies are required for capturing, isolating and subsequently analyzing single cells in a high throughput context. Within the MeBioS Biosensor group, an innovative approach for conducting high throughput time-lapse studies on single non-adherent cells has been recently demonstrated, based on digital microfluidic platform (DMF). In the current work, we present an application of the adapted system, expanded with optical-tweezers3, for capturing and releasing magnetic bead-coupled single bacterial cells from a microwell. The microfluidic chip consists of (i) a grounding plate containing an array of 62,500 microwells (4 µm wide and 3 µm deep) and (ii) an actuation plate containing an array of electrodes. For obtaining suitable bacteria capturing efficiency, tosyl-activated superparamagnetic beads were conjugated with anti-Salmonella Typhimurium IgG1 at different antibody concentrations. By performing automated actuations, the microwells were seeded with antibody-functionalized-superparamagnetic beads, followed by an on-chip incubation of the beads with Salmonella cells. The array was then washed five times to remove unbound bacteria. A bacteria capturing efficiency of 48 % was obtained for an antibody concentration of 0.24 µg/µl. To test the specificity of bacteria capturing, control samples with no-antibody-conjugated superparamagnetic beads were tested, which resulted in a capturing efficiency of less than 0.04%. For retrieving bacteria bound magnetic beads, an IR optical-tweezers setup was built and the optimal buffer condition was determined for optically levitating the beads. As a proof of concept, bacteria bound to magnetic beads were optically retrieved from one microwell, transported and seeded in another empty microwell. In conclusion, we demonstrate for the first time, a DMF platform enabled spatial isolation and selective retrieval of single non-adhering cells bound to magnetic beads using optical tweezers. Research focused on screening and selection of single cells within a large population, and identification of their genetic makeup is addressed with this novel digital microfluidic platform.status: publishe

Digital microfludic chip technology for osmotic potential studies on single cells

Analysis of single cells that are naturally non-adhering to a substrate is still hindered by the scarce availability of systems for precise manipulation and retention of cells at a desired location [1]. Some types of experiments, such as osmotic potential studies, can be conducted at the single cell level using micropipettes or micromanipulators but with extremely low throughput as only one cell can be handled during each analysis [2]. Here we demonstrate the potential of EWOD-based DMF chips as an efficient and valuable alternative for the analysis, at a single cell resolution, of a collection of cells at once. We describe a novel application of electrowetting-on-dielectric (EWOD) based digital microfluidic (DMF) chips for osmotic potential studies of individual protoplast cells isolated from Arabidopsis thaliana leaves. A collection of cells is immobilized on the chip surface through conjugation to superparamagnetic microparticles and the use of an external magnet. The DMF chip allows for automatic and simultaneous treatment of the cells with different osmotic conditions while volumetric changes are monitored with an external camera. The water permeability coefficients (Pos) of single cells are extrapolated from the measured volumetric changes. The Pos value correspond to the values reported in literature and those obtained with the traditional analysis systems. This work demonstrates how EWOD-based DMF chips can boost the throughput of osmotic potential studies by allowing the analysis of a collection of single protoplasts at once. Digital microfluidics chips are fabricated with standard lithography techniques as previously described [3]. To allow monitoring of the swelling or shrinking of protoplasts upon osmotic treatment, transparent visualization windows are inserted in the actuation electrodes (Fig.1). The setup used in our group is illustrated in Fig.1. Protoplasts are functionalized with biotinylated-concanavalin A and conjugated to streptavidin coated magnetic beads (Fig. 2). Cells are retained onto the chip surface through an external magnetic field and exposed to different osmotic conditions in an automated way. We demonstrate the applicability of this platform for single cell studies by successfully immobilizing a group of protoplasts on the chip surface and analyzing the volumetric change of each single cell after an automated exposure to different osmotic conditions. The changes in the volume of protoplasts are monitored as function of time after applying a change in osmolarity. Osmotic water permeability coefficient (Pos) values of single protoplasts are extrapolated from the measured volumetric changes over time using the initial rate method [4] (Fig 3). Compared to traditional studies that use micropipettes to hold one cell of interest, our system allows for a substantial increase of the number of measured cells ranging between one and ten per analysis.status: publishe

Digital Microfluidics for Single Bacteria Capture and Selective Retrieval Using Optical Tweezers

When screening microbial populations or consortia for interesting cells, their selective retrieval for further study can be of great interest. To this end, traditional fluorescence activated cell sorting (FACS) and optical tweezers (OT) enabled methods have typically been used. However, the former, although allowing cell sorting, fails to track dynamic cell behavior, while the latter has been limited to complex channel-based microfluidic platforms. In this study, digital microfluidics (DMF) was integrated with OT for selective trapping, relocation, and further proliferation of single bacterial cells, while offering continuous imaging of cells to evaluate dynamic cell behavior. To enable this, magnetic beads coated with Salmonella Typhimurium-targeting antibodies were seeded in the microwell array of the DMF platform, and used to capture single cells of a fluorescent S. Typhimurium population. Next, OT were used to select a bead with a bacterium of interest, based on its fluorescent expression, and to relocate this bead to a different microwell on the same or different array. Using an agar patch affixed on top, the relocated bacterium was subsequently allowed to proliferate. Our OT-integrated DMF platform thus successfully enabled selective trapping, retrieval, relocation, and proliferation of bacteria of interest at single-cell level, thereby enabling their downstream analysis

Digital microfluidics for cytotoxicity studies on yeast cells at spatio-temporal resolution

Elucidating the heterogeneous responses of single cells to external perturbation is one of the central interests in cell biology. However, continuous monitoring of non-adhering single cells is still hindered by the limited availability of systems for precise manipulation and retention of cells at a desired location. Flow cytometry is one of the most widely used techniques to characterize non-adhering cells, however, its inability to follow the response of single cells in time is a major drawback1. Here we describe an application of electrowetting-on-dielectric (EWOD) based digital microfluidic (DMF) chip2, as a platform for trapping of non-adhering cells in an array of microwells. Consequently, the single cell response to a drug incubation can be monitored with a spatio-temporal resolution in an automatic way. The scheme of DMF device for trapping single yeast cells (Saccharomyces cerevisiae) is illustrated in Fig.1. The device consists of two parallel glass plates, namely the actuation plate and grounding plate (Fig.1a). An array consisting of 22000 microwells (5 µm wide and 3 µm deep) was fabricated in the Teflon layer of the grounding plate3. The microwells have hydrophilic bottom and hydrophobic sidewalls (Fig.1a,inset). In order to trap yeast cells, the cell droplet was transported over the array for multiple times, referred to as seeding cycles (Fig.1a), resulting in entrapment of single cells in femtoliter droplets in the microwells. These spatially confined cells were then subjected to antifungal treatments (Fig.1b) and the viability of single cells using the viability dye Propidium Iodide (PI) was monitored with time-lapse microscopy (Fig.1c). The cell’s response upon treatment with various concentrations of amphotericin B (AmB), a cell membrane permeabilizing antifungal drug that also induces programmed cell death at certain doses4, was studied over a period of 6 hours during which viability of the cells was monitored every 15 minutes. We found a dose-dependent increase of the number of PI-positive cells (dead cells) over time. The maximum number of dead cells for each AmB dose was reached after approx. 270 min of incubation (Fig. 2). Treating the cells with higher AmB doses (>50 µM) did not result in 100 % PI-positive staining of cells. Only upon UV treatment, all cells stained PI-positive, indicating complete cell death of the culture. This result indicates that a subpopulation of the yeast culture treated with AmB is PI-negative, indicative for increased AmB tolerance or programmed cell death. Cells dying via programmed cell death or apoptosis are typically characterized by PI-negative staining as the cell membrane is still intact. In conclusion, we demonstrate for the first time, an EWOD based DMF device-enabling spatial isolation of single non-adhering yeast cells and their subsequent cytotoxicity read-out upon drug treatment. Our system allows efficient monitoring of cellular response over a period of time. Research focused on elucidating kinetics of cell responses to drug stimuli and screening for novel antifungals is addressed with this novel digital microfluidic platform.(*Shared first co-authors)status: publishe

Digital microfluidic chip for implementing time-resolved cell based studies

Single cell analysis (SCA) has obtained great importance among biologists for elucidating cell-to-cell differences that are difficult to recognize in bulk measurements. Flow cytometry is one of the most widely used techniques to characterize non-adhering cells, however, its inability to analyze cells with spatio-temporal resolution is a major drawback. Continuous monitoring and analysis of non-adhering cells requires involvement of certain trapping systems for the precise manipulation and retention of these cells at a desired location. To date, various microfluidic platforms have demonstrated high-throughput single cell studies of non-adhering cells by implementing cell-specific trapping strategies1,2. In this study, we demonstrate the potential of electro-wetting-on-dielectric based digital microfluidics (EWOD-DMF), as automated and miniaturized systems to trap single yeast cells and analyze their responses towards antifungal treatment at a single cell resolution over time. A DMF chip is a two-plate system in which the bottom plate carries a planar array of chromium electrodes (2.8 mm x 2.8 mm) on a glass substrate, covered with a layer of dielectric material and Teflon-AF, and the top plate consists of an Aluminum-Teflon coated glass plate (Fig.1(a,b)). Discrete reagent droplets sandwiched between the two plates are transported across the electrode array based on the electrowetting-on-dielectric (EWOD) actuation principle. In addition, a microarray containing 21,000 cylindrical cavities (5 μm diameter and 3 μm deep) is fabricated in the top plate Teflon layer in a such way that the walls of the cavity remain hydrophobic whereas, the cavity bottom is hydrophilic. The on-chip cell trapping is achieved by allowing the cells to sediment on the array for 10 minutes. This is followed by subsequently transporting the cell droplet over the microarray multiple times (referred to as transport cycles). During this time, the cells enter the microwells by capillary forces and are trapped in the hydrophilic region of the cavities (Fig. 1(c)). The trapped cells are then subjected to antifungal treatment (Amphotericin B; AMB) and monitored over a period of 4 hours. The effect of the number of transport cycles on cell trapping efficiency and cell viability was analyzed using propidium iodide. Out of the three different transport cycles tested, the optimum number of transport cycles was obtained as 15 (Fig.2(a)). The decrease in trapping efficiency observed at higher transport cycles, indicate the removal of previously trapped cells and is possibly due to the strong shear forces that originated from transporting the cell droplet multiple times. In line with this hypothesis, a decrease in the viability of trapped cells was observed while increasing the number of transport cycles. Secondly, the response of trapped cells towards treatment with 100 μM AMB were investigated. Time-dependent killing was observed and approximately 100 % cell death was reached after 4 hours (Fig. 2(b)). To the best of our knowledge, this is the first such demonstration of the use of EWOD-based DMF chips for in vitro cytotoxicity assays on single yeast cells with spatio-temporal resolution. More importantly, this platform can be used for thorough screening of newly developed antifungals in a semi high-throughput manner.status: publishe