Flu is caused by the influenza virus that, due to mutations, keeps our body vulnerable for infections, making early diagnosis essential. Although immuno-based diagnostic tests are available, they have low sensitivity and reproducibility. In this paper, the prospect of detecting influenza A virus using digital ELISA has been studied. To appropriately select bioreceptors for this bioassay, seven commercial antibodies against influenza A nucleoprotein were methodically tested for their reactivity and binding affinity. The study has been performed on two markedly different platforms, being an enzyme-linked immunosorbent assay and a surface plasmon resonance system. The selected antibodies displayed completely different behavior on the two platforms and in various assay configurations. Surprisingly, the antibodies that showed overall good reactivity on both platforms had the highest dissociation constant among the tested antibodies, suggesting that, although important, binding affinity is not the only parameter to be considered when selecting antibodies. Moreover, only one antibody had the capacity to capture the nucleoprotein directly in lysis buffer used for releasing this viral protein, which might pose a huge advantage when developing assays with a fast time-to-result. This antibody was implemented on an in-house developed digital ELISA platform for ultrasensitive detection of recombinant nucleoprotein, reaching a detection limit of 4 ± 1 fM in buffer and 10 ± 2 fM in 10-fold diluted nasopharyngeal swabs, which is comparable to currently available fast molecular detection techniques. These results point to a great potential for ultrasensitive immuno-based influenza detection.
Single cell analysis (SCA) has gained increased popularity for elucidating cellular heterogeneity at genomic, proteomic and cellular levels. Flow cytometry is considered as one of the most widely used techniques to characterize single cell responses; however, its inability to analyse cells with spatio-temporal resolution poses a major drawback. Here, we introduce a digital microfluidic (DMF) platform as a useful tool for conducting studies on isolated yeast cells in a high-throughput fashion. The reported system exhibits (i) a microwell array for trapping single non-adherent cells by shuttling a cell-containing droplet over the array, and allows (ii) implementation of high-throughput cytotoxicity assays with enhanced spatio-temporal resolution. The system was tested for five different concentrations of the antifungal drug Amphotericin B, and the cell responses were monitored over time by time lapse fluorescence microscopy. The DMF platform was validated by bulk experiments, which mimicked the DMF experimental design. A correlation analysis revealed that the results obtained on the DMF platform are not significantly different from those obtained in bulk; hence, the DMF platform can be used as a tool to perform SCA on non-adherent cells, with spatio-temporal resolution. In addition, no external forces, other than the physical forces generated by moving the droplet, were used to capture single cells, thereby avoiding cell damage. As such, the information on cellular behaviour during treatment could be obtained for every single cell over time making this platform noteworthy in the field of SCA.
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.
Dynamic follow up and analysis of isolated single cells that display non-adhering behaviour is hindered by the fact that they float in the suspension medium and thus requires the implementation of systems that physically entrap the cells for successful analysis. We describe the potential of digital microfluidic (DMF) chip technology for conducting analysis of cells in suspension at the single cell resolution. More specifically, we demonstrate the use of DMF technology for the analysis of a group of single individual protoplasts from Arabidopsis thaliana plants that are labelled with magnetic particles and are immobilized on the DMF chip by magnetic forces. By transporting droplets with different osmotic conditions to the site where the cells are trapped, we challenge the cells and monitor their responses dynamically with a camera. The use of DMF technology for performing water potential measurements has the following advantages: (i) solid particles such as cells and magnetic beads are manipulated on the chip without the risk of clogging channels, (ii) low shear stress during droplet unit operations like mixing and transport that characterize DMF are particularly suited for analysis of delicate cells types that lack cell walls such as protoplasts, (iii) the throughput of the analysis is strongly increased as multiple protoplasts are analysed simultaneously, in contrast with the traditional methods that can handle and challenge only one cell at a time. We show that the DMF analysis platform is effective in creating the steep osmotic gradients required for calculation of water permeability coefficients (P) as the values found are comparable with previously reported ones thus validating the suitability of the technology for such studies. This work illustrates a proof of concept for the applicability of DMF as an unparalleled and promising system for implementing single cells studies on non-adhering cells in an automated way.
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.
Digital microfluidics has emerged in the last years as a promising liquid handling technology for a variety of applications. Here, we describe in detail how to build up an electrowetting-on-dielectric-based digital microfluidic chip with unique advantages for performing single-molecule detection. We illustrate how superparamagnetic particles can be printed with very high loading efficiency (over 98 %) and single-particle resolution in the microwell array patterned in the Teflon-AF surface of the grounding plate of the chip. Finally, the potential of the device for its application to single-molecule detection is demonstrated by the ultrasensitive detection of the biotinylated enzyme β-Galactosidase captured on streptavidin-coated particles in the described platform.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.