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Sample-to-answer automation of multiplexed bioassay panels represents a key selling point of microfluidic Lab-on-a-chip devices. For real-world point-of-use scenarios, on-chip reagent storage is a key requirement for enabling the downstream microfluidic operations (e.g., resuspension of dried or lyophilized reagents using stored buffers) . Several methods have been developed to this end with issues around cost, manufacturability and reliability being the key factors [2, 3]. This paper reports manufacture, assembly and characterization of a novel, low-cost and scalable technology for reagent storage and release based on sacrificial barrier films. We demonstrate an average evaporative volume loss of stored DI water at room temperature (21 o C) of 0.38% (± 0.3) for 42 days at a significantly low current materials cost of 30 cents per unit. The outlet of a chip-based reagent-storage chamber is transiently blocked by a barrier film composed of a distinct fluoropolymer on pressure sensitive adhesive. Due to its excellent hydrophobic, biocompatible and water / vapor barrier properties, the film allows for minimization of evaporation related losses of aqueous reagents. An immiscible and biocompatible oil-based liquid is layered on top of the aqueous phase. During storage and shipping, laminar conditions prevent the oil phase from reaching the barrier film. Under rotationally induced artificial gravity, the denser ancillary liquid displaces the reagent to contact the sacrificial film (Figure 1). It then retained in a volume-matched capture chamber so that the aqueous reagent is isolated further downstream. We demonstrate the ability of this actitation mechanism within a band of ±1.40 Hz for varying radial positions and release frequencies. Key characteristics of the reagent storage technology (Figure 2): a) Average evaporative losses of 0.38% (± 0.3) (test units with 500 µl DI water) when stored at room temperature (21 o C) for 42 days. b) Ability of the reagent storage units to handle real time transport was tested by air + ground shipping of 31 individual units on 6 discs over 10 days in transit sent in 3 different packages. No significant losses (when compared to unshipped units) in either the final amount of reagent released post-transport or the release frequencies were observed. c) Cumulative systemic losses of reagents post release within the microfluidic chips were 2.5µl (± 1.3 µl). All the release liquid is isolated after release in the capture chamber thus delivering only the aqueous reagent to the final chamber. d) Current cost of a barrier film insert is ~ €0.30. e) Centrifugal release frequency bands are characterized as a function of radial position and the depth of the upper and lower parts of the dual depth channel (Fig 2 B, C, D). Word Count: 490 References: 1. Smith et al. CD-Based Microfluidics for Primary Care in Extreme Point-of-Care Settings. Micromachines 2016, 7(2), 22. 2. Oordt et al. Miniature stick-packaging-an industrial technology for pre-storage and release of reagents in lab on-a-chip systems. Lab Chip, 2013,13, 2888-2892. 3. Smith et al. Blister pouches for effective reagent storage on microfluidic chips for blood cell counting.
Most point-of-care applications require onboard storage of liquid reagents such as buffers and stains. The reliable handling and release of these reagents is key to enabling the multiple unit operations on these platforms. We present here a centrifugal buoyancy-driven release mechanism , where the balance between a stored aqueous reagent (to protect a lipophilic dissolvable-film (DF)) and an ancillary oil liquid allows for controlled release. At the beginning of the assay protocol, the disc is spun to generate sufficient centrifugal pressure allowing the denser ancillary phase to reliably displace the aqueous phase to thus open the DF, due to stratification of the immiscible phases.
It is commonly recognised that microfluidic technologies will leverage a broad range of decentralised "point-of-use" applications in areas such as healthcare, pharma, veterinary medicine, agrifood and life-science research as well as monitoring of the environment, infrastructures and industrial processes. However, in particular during market entry, many of these mostly life-science related technologies will only reach comparatively small production numbers, thus preventing economy-of-scale effects to assure quick recovery sunk cost for development of product and production technologies. This presentation illustrates our path from idea to robust microfluidic design and seamless scale-up of manufacture from prototyping to larger test series for the example of the centrifugal-microfluidic technologies. This "Lab-on-a-Disc" platform can be flexibly configured to implement a wide repertoire of fluidically homologous liquid handling protocols at the backbone general chemistry, immunoassays, nucleic acid testing and cell analysis. Our quality-by-design approach thus substantially expedites and de-risks development of many microfluidics-enabled solutions towards high-technology-readiness levels.
The commercial development of many microfluidics-enabled commercial devices requires rapid optimization and cost-efficient development of competitively priced, and still very rugged, mostly single-use devices. A key challenge remains the seamless scale-up of manufacture from laborious prototyping techniques based on low-throughput machinery and frequently expensive materials to pilot series and industrializable (polymer) mass replication, (bio-)functionalization and assembly schemes. Furthermore, the time-scales and budgets involved from idea to product are often hard to accept for investors, in particular when considering that many microfluidics-based products will, at least initially, be restricted to comparatively small niche markets or rather tiny fractions of larger markets; hence, economy-of-scale effects are difficult to reach for re-covering (sunk) for research and technology development as well as set up of manufacture. -- This paper presents a platform approach adopted from many mature industries like automotive or microelectronics for assuring cost-efficient, significantly expedited development of process inte-grated and automated, microfluidics-enabled solutions at high tech-nology readiness levels (TRLs) with typical application in decentra-lised "point-of-use" handling and testing of biosamples. Key para-digms supporting this platform approach towards industrialization of microfluidics-enabled solutions are design-for-manufacture (DfM) for seamless scale-up from prototyping to production, quality-by-design (QbD) for robust operation and readiness for scale-up (RfS) towards mass fabrication.
Integrated microfluidic technologies have demonstrated significant benefits for a range of applications, mostly in the context of decentralised bioanalytical multiparameter testing at the point of use with primary applications in healthcare, pharma, veterinary medicine, agrifood and life-science research as well as monitoring of the environment, infrastructures and industrial processes. To decisively accelerate development, FPC@DCU , the Fraunhofer Project Centre for Embedded Bioanalytical Systems at Dublin City University, pursues a coherent, design-for-manufacture (DfM) themed platform approach ; new, microfluidics-enabled sample-to-answer solutions are generated from a common library of geometrically parametrized fluidic elements for flow control, e.g. valves and routers, coordinating sequential and parallelized liquid handling protocols comprising of mixing, metering, aliquoting, reagent storage and particle removal steps. The impact of manufacturing tolerances and artefacts on functionality is considered along all stages of scale-up, ranging from prototyping by ultraprecision milling to tooling, polymer repliation (e.g. by injection molding), biofunctionalization and automated assembly of multi-component systems. DfM thus assures seamless scale-up from pilot series for fluidic design optimization to bioassay development and large-scale production, thus substantially supporting regu-latory approval and significantly de-risking commercial product development. This presentation will illustrate the design-for-manufacture strategy to reach high technology-readiness levels along the example of FPC@DCU's centrifugal microfluidic "Lab-on-a-Disc" (LoaD) platform.
This paper reports centrifugal microfluidic automation of surface plasmon resonance (SPR) detection of a protein concentration series (IgG). Using event-triggered rotational flow control based on water-dissolvable film (DF) valving, blood separation, metering, incubation and washing steps were parallelized on an integrated "Lab-on-a-Disc" (LoaD) cartridge. In a prism-based coupling scheme, the change in the angle of minimum reflectivity due to specific biomolecular binding was measured by a conventional smartphone camera.
Excessive cell division is characteristic for the onset of cancer. By removing the enabling cell spindle, many chemotherapeutic agents interfere with this key process also referred to mitosis. However, cancer cells vary widely in their susceptibility to these cytotoxic agents. It has been demonstrated that cells are significantly less deformable during mitosis than during interphase during their cycle . This work presents a microfluidic tool for early-stage identification of cell spindle resistance to chemotherapy based on cell stiffness to avoid ineffective treatment and relapse.
HUVEC and EA.hy926 are cell models acting as key reporters for staging patients with cardiovascular disease (CVD). For the first time, microfluidic and optical technologies are integrated to resolve the real-time response of individual cell types in a population upon stimulation with the specific, inflammatory agents, tumor necrosis factor alpha (TNF-α) and lipopolysaccharide (LPS). The emerging expression markers PECAM-1 (CD31), VCAM-1 (CD106), ICAM-1 (CD54) and E-Selectin (CD62E) indicate the onset of early stage endothelial cell dysfunction, a key characteristic in the diagnosis of CVD. Current approaches for evaluation involve assessment of endothelial dependent vasodilation in vivo and / or non-invasive methods with low resolution or high variability. In the present study, we investigated detection of endothelial dysfunction with a novel, single-cell optical multi-parameter monitoring system. EXPERIMENTAL Human umbilical vein endothelial cells (HUVECs) were cultured as per standard cell culture protocol and treatments of TNF-α (20 ng ml-1 for 24 hours) and LPS (20 ng ml-1 for 24 hours) were applied for inducing inflammation. The treated cells, along with a negative control population (untreated HUVECs), are individually captured at single-cell occupancy distribution in scale-matched traps introduced in the sedimentation path of a centrifugal microfluidic chip . Once arrayed in these 'V-cups', the cells are stained and incubated with the relevant antibody. Fluorescence from the residual endothelial expression markers are acquired by instrument-mounted optical modules (Figs. 1-3). RESULTS AND DISCUSSION Expressed levels of PECAM-1, ICAM-1 and E-selectin all elevated under inflammatory conditions, whilst the levels of VCAM-1 tended to decrease. These results were then benchmarked against gold-standard methods and flow cytometry detection; similar patterns were observed across all four markers in each cell condition (Fig. 4). Replicate experiments performed on EA.hy926 cells, both, on chip (Fig. 5) and by flow cytometry (Fig. 6), also showed similar expression levels and patterns across all four markers for each of the three cell conditions. Our opto-fluidic system measures a specific and unique fluorescence signal for each endothelial expression marker investigated under three different cell conditions, across two endothelial cell models. The activation levels, which can be classified at single-cell resolution, prove to be indicative for endothelial cell dysfunction, an early stage indicator of the onset of CVD. Compared to classical in-vivo studies, the novel system enables rapid analysis, reduces precious sample volume and facilitates sample preparation. Acknowledgements: The research was conducted with the financial support of Science Foundation Ireland (SFI) and Fraunhofer-Gesellschaft under the SFI Strategic Partnership Programme [grant number 16/SPP/3321].
This work shows for the first time how unavoidable tolerances in manufacturing and experimental input parameters have a decisive influence on the reliability of flow control in Lab-on-a-Disk (LoaD) platforms and must therefore be considered towards larger scale fluidic integration (LSI). INTRODUCTION With their increasing commercialization, assuring operational robustness under economically viable manufacturing schemes becomes paramount for microfluidic Lab-on-a-Chip systems. With the example of centrifugo-pneumatic Lab-on-a-Disk (LoaD) platform controlled by dissolvable-film (DF) valves [1, 2], this work illustrates for the first time how unavoidable tolerances in manufacturing and experimental input parameters such as channel dimensions, contact angles, liquid properties and environmental conditions critically affect the reliability of flow control, and thus need to be imperatively addressed towards larger-scale fluidic integration (LSI).
Cellular activation and inflammation leading to endothelial dysfunction is associated with cardiovascular disease (CVD). We investigated whether a single cell label-free multi parameter optical interrogation system can detect endothelial cell and endothelial progenitor cell (EPC) activation in vitro and ex vivo, respectively. Cultured human endothelial cells were exposed to increasing concentrations of tumour necrosis factor alpha (TNF-α) or lipopolysaccharide (LPS) before endothelial activation was validated using fluorescence-activated cell sorting (FACS) analysis of inflammatory marker expression (PECAM-1, E-selectin and ICAM-1). A centrifugal microfluidic system and V-cup array was used to capture individual cells before optical measurement of light scattering, immunocytofluorescence, auto-fluorescence (AF) and cell morphology was determined. In vitro, TNF-α promoted specific changes to the refractive index and cell morphology of individual cells concomitant with enhanced photon activity of fluorescently labelled inflammatory markers and increased auto-fluorescence (AF) intensity at three different wavelengths, an effect blocked by inhibition of downstream signalling with Iκβ. Ex vivo, there was a significant increase in EPC number and AF intensity of individual EPCs from CVD patients concomitant with enhanced PECAM-1 expression when compared to normal controls. This novel label-free ‘lab on a disc’ (LoaD) platform can successfully detect endothelial activation in response to inflammatory stimuli in vitro and ex vivo.