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Transplantable ready-made microvessels have therapeutic potential for tissue regeneration and cell replacement therapy. Inspired by the natural rapid angiogenic sprouting of microvessels in vivo, engineered injectable 3D microvessel networks are created using thermoplastic elastomer (TPE) microfluidic devices. The TPE material used here is flexible, optically transparent, and can be robustly yet reversibly bonded to a variety of plastic substrates, making it a versatile choice for microfluidic device fabrication because it overcomes the weak self-adhesion properties and limited manufacturing options of poly(dimethylsiloxane) (PDMS). By leveraging the reversible bonding characteristics of TPE material templates, we present their utility as an organ-on-a-chip platform for forming and handling microvessel networks, and demonstrate their potential for animal-free tissue generation and transplantation in clinical applications. We first show that TPE-based devices have nearly 6-fold higher bonding strength during the cell culture step compared to PDMS-based devices while simultaneously maintaining a full reversible bond to (PS) culture plates, which are widely used for biological cell studies. We also demonstrate the successful generation of perfusable and interconnected 3D microvessel networks using TPE–PS microfluidic devices on both single and multi-vessel loading platforms. Importantly, after removing the TPE slab, microvessel networks remain intact on the PS substrate without any structural damage and can be effectively harvested following gel digestion. The TPE-based organ-on-a-chip platform offers substantial advantages by facilitating the harvesting procedure and maintaining the integrity of microfluidic-engineered microvessels for transplant. To the best of our knowledge, our TPE-based reversible bonding approach marks the first confirmation of successful retrieval of organ-specific vessel segments from the reversibly-bonded TPE microfluidic platform. We anticipate that the method will find applications in organ-on-a-chip and microphysiological system research, particularly in tissue analysis and vessel engraftment, where flexible and reversible bonding can be utilized. [ABSTRACT FROM AUTHOR]

Droplet digital polymerase chain reaction (ddPCR) stands out as a highly sensitive diagnostic technique that is gaining traction in infectious disease diagnostics due to its ability to quantitate very low numbers of viral gene copies. By partitioning the sample into thousands of droplets, ddPCR enables precise and absolute quantification without relying on a standard curve. However, current ddPCR systems often exhibit relatively low levels of integration, and the analytical process remains dependent on elaborate workflows for up-front sample preparation. Here, we introduce a fully-integrated system seamlessly combining viral lysis, RNA extraction, emulsification, reverse transcription (RT) ddPCR, and fluorescence readout in a sample-to-answer format. The system comprises a disposable microfluidic cartridge housing buffers and reagents required for the assay, and a centrifugal platform that allows for pneumatic actuation of liquids during rotation, enabling automation of the workflow. Highly monodisperse droplets (∼50 μm in diameter) are produced using centrifugal step emulsification and automatically transferred to an integrated heating module for target amplification. The platform is equipped with a miniature fluorescence imaging system enabling on-chip read-out of droplets after RT-ddPCR. We demonstrate sample-to-answer detection of SARS-CoV-2 N and E genes, along with RNase P endogenous reference, using hydrolysis probes and multiplexed amplification within single droplets for concentrations as low as 0.1 copy per μL. We also tested 14 nasopharyngeal swab specimens from patients and were able to distinguish positive and negative SARS-CoV-2 samples with 100% accuracy, surpassing results obtained by conventional real-time amplification. [ABSTRACT FROM AUTHOR]

Freestanding, flexible and open through-hole polymeric micro- and nanostructured membranes were successfully fabricated over large areas (>16 cm2) via solvent removal of sacrificial scaffolds filled with polymer resin by spontaneous capillary flow. Most of the polymeric membranes were obtained through a rapid UV curing processes via cationic or free radical UV polymerisation. Free standing microstructured membranes were fabricated across a range of curable polymer materials, including: EBECRYL3708 (radical UV polymerisation), CUVR1534 (cationic UV polymerisation) UV lacquer, fluorinated perfluoropolyether urethane methacrylate UV resin (MD700), optical adhesive UV resin with high refractive index (NOA84) and medical adhesive UV resin (1161-M). The present method was also extended to make a thermal set polydimethylsiloxane (PDMS) membranes. The pore sizes for the as-fabricated membranes ranged from 100 µm down to 200 nm and membrane thickness could be varied from 100 µm down to 10 µm. Aspect ratios as high as 16.7 were achieved for the 100 µm thick membranes for pore diameters of approximately 6 µm. Wide-area and uniform, open through-hole 30 µm thick membranes with 15 µm pore size were fabricated over 44 × 44 mm2 areas. As an application example, arrays of Au nanodots and Pd nanodots, as small as 130 nm, were deposited on Si substrates using a nanoaperture polymer through-hole membrane as a stencil. [ABSTRACT FROM AUTHOR]

Testing for SARS-CoV-2 is one of the most important assets in COVID-19 management and mitigation. At the onset of the pandemic, SARS-CoV-2 testing was uniquely performed in central laboratories using RT-qPCR. RT-qPCR relies on trained personnel operating complex instrumentation, while time-to-result can be lengthy (e.g., 24 to 72 h). Now, two years into the pandemic, with the surge in cases driven by the highly transmissible Omicron variant, COVID-19 testing capabilities have been stretched to their limit worldwide. Rapid antigen tests are playing an increasingly important role in quelling outbreaks by expanding testing capacity outside the realm of clinical laboratories. These tests can be deployed in settings where repeat and rapid testing is essential, but they often come at the expense of limited accuracy and sensitivity. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) provides a number of advantages to SARS-CoV-2 testing in standard laboratories and at the point-of-need. In contrast to RT-qPCR, RT-LAMP is performed at a constant temperature, which circumvents the need for thermal cycling and translates into a shorter analysis time (e.g., <1 h). In addition, RT-LAMP is compatible with colorimetric detection, facilitating visualization and read-out. However, even with these benefits, RT-LAMP is not yet clinically deployed at its full capacity. Lack of automation and integration of sample preparation, such as RNA extraction, limits the sensitivity and specificity of the method. Furthermore, the need for cold storage of reagents complicates its use at the point of need. The developments presented in this work address these limitations: We describe a fully automated SARS-CoV-2 detection method using RT-LAMP, which also includes up-front lysis and extraction of viral RNA, performed on a centrifugal platform with active pneumatic pumping, a disposable, all-polymer-based microfluidic cartridge and lyophilized reagents. We demonstrate that the limit of detection of the RT-LAMP assay itself is 0.2 copies per μL using N and E genes as target sequences. When combined with integrated RNA extraction, the assay sensitivity is 0.5 copies per μL, which is highly competitive to RT-qPCR. We tested the automated assay using 12 clinical swab specimens from patients and were able to distinguish positive and negative samples for SARS-CoV-2 within 60 min, thereby obtaining 100% agreement with RT-qPCR results. [ABSTRACT FROM AUTHOR]

We investigate the formation of suspended magnetic nanoparticle (MNP) assemblies (M-clouds) and their use for in situ bacterial capture and DNA extraction. M-clouds are obtained as a result of magnetic field density variations when magnetizing an array of micropillars coated with a soft ferromagnetic NiP layer. Numerical simulations suggest that the gradient in the magnetic field created by the pillars is four orders of magnitude higher than the gradient generated by the external magnets. The pillars therefore serve as the sole magnetic capture sites for MNPs which accumulate on opposite sides of each pillar facing the magnets. Composed of loosely aggregated MNPs, the M-cloud can serve as a porous capture matrix for target analyte flowing through the array. The concept is demonstrated by using a multifunctional M-cloud comprising immunomagnetic NPs (iMNPs) for capture of Escherichia coli O157:H7 from river water along with silica-coated NPs for subsequent isolation and purification of microbial DNA released upon bacterial lysis. Confocal microscopy imaging of fluorescently labeled iMNPs and E. coli O157:H7 reveals that bacteria are trapped in the M-cloud region between micropillars. Quantitative assessment of in situ bacterial capture, lysis and DNA isolation using real-time polymerase chain reaction shows linear correlation between DNA output and input bacteria concentration, making it possible to confirm E. coli 0157:H7 at 103 cells per mL. The M-cloud method further provides one order of magnitude higher DNA output concentrations than incubation of the sample with iMNPs in a tube for an equivalent period of time (e.g., 10 min). Results from assays performed in the presence of Listeria monocytogenes (at 106 cells per mL each) suggest that non-target organisms do not affect on-chip E. coli capture, DNA extraction efficiency and quality of the eluted sample. [ABSTRACT FROM AUTHOR]

Fractionating whole blood and separating its constituent components one from another is an essential step in many clinical applications. Currently blood sample handling and fractionation processes remain a predominantly manual task that require well-trained operators to produce reliable and reproducible results. Herein, we demonstrate an advanced on-chip whole human blood fractionation and cell isolation process combining (i) an aqueous two-phase system (ATPS) to create complex separation layers with (ii) a centrifugal microfluidic platform (PowerBlade) with active pneumatic pumping to control and automate the assay. We use a polyethylene glycol (PEG) and dextran (DEX) mixture as the two-phase density gradient media and our automated centrifugal microfluidic platform to fractionate blood samples. Different densities of precisely tuned PEG–DEX solutions were tested to match each of the cell types typically targeted during blood fractionation applications. By employing specially designed microfluidic devices, we demonstrate the automation of the following steps: loading of a whole blood sample on-chip, layering of the blood on the ATPS solution, blood fractionation, precise radial repositioning of the fractionated layers, and finally extraction of multiple, selected fractionated components. Fractionation of up to six distinct layers is shown: platelet-rich plasma, buffy coat, PEG, DEX with neutrophils, red blood cells (RBCs) and high density gradient media (HDGM). Furthermore, through controlled dispensing of HDGM to the fractionation chamber, we show that each of the fractionated layers can be repositioned radially, on-the-fly, without disturbing the interfaces, allowing precise transfer of target fractions and cell types into external vials via a chip-to-world interface. Cell counting analysis and cell viability studies showed equivalence to traditional, manual methods. An overall cell viability greater than 90% of extracted cells demonstrates that the proposed approach is suitable for cell isolation applications. This proof-of-principle demonstration highlights the utility of the proposed system for automated whole blood fractionation and isolation for blood cell applications. We anticipate that the proposed approach will be a useful tool for many clinical applications such as standard cell isolation procedures and other bioanalytical assays (e.g., circulating tumor cells, and cell and gene therapy). [ABSTRACT FROM AUTHOR]

We uncover anisotropic permeability in microfluidic deterministic lateral displacement (DLD) arrays. A DLD array can achieve high-resolution bimodal size-based separation of microparticles, including bioparticles, such as cells. For an application with a given separation size, correct device operation requires that the flow remains at a fixed angle to the obstacle array. We demonstrate via experiments and lattice-Boltzmann simulations that subtle array design features cause anisotropic permeability. Anisotropic permeability indicates the microfluidic array's intrinsic tendency to induce an undesired lateral pressure gradient. This can cause an inclined flow and therefore local changes in the critical separation size. Thus, particle trajectories can become unpredictable and the device useless for the desired separation task. Anisotropy becomes severe for arrays with unequal axial and lateral gaps between obstacle posts and highly asymmetric post shapes. Furthermore, of the two equivalent array layouts employed with the DLD, the rotated-square layout does not display intrinsic anisotropy. We therefore recommend this layout over the easier-to-implement parallelogram layout. We provide additional guidelines for avoiding adverse effects of anisotropy on the DLD. [ABSTRACT FROM AUTHOR]

Detecting pathogenic bacteria in food or other biological samples with lab-on-a-chip (LOC) devices requires several sample preparation steps prior to analysis which commonly involves cleaning complex sample matrices of large debris. This often underestimated step is important to prevent these larger particles from clogging devices and to preserve initial concentrations when LOC techniques are used to concentrate or isolate smaller target microorganisms for downstream analysis. In this context, we developed a novel microfluidic system for membrane-free cleaning of biological samples from debris particles by combining hydrodynamic focusing and inertial lateral migration effects. The microfluidic device is fabricated using thermoplastic elastomers being compatible with thermoforming fabrication techniques leading to low-cost single-use devices. Microfluidic chip design and pumping protocols are optimized by investigating diffusive losses numerically with coupled Navier-Stokes and convective-diffusion theoretical models. Stability of inertial lateral migration and separation of debris is assessed through fluorescence microscopy measurements with labelled particles serving as a model system. Efficiency of debris cleaning is experimentally investigated by monitoring microchip outlets with in situ optical turbidity sensors, while retention of targeted pathogens (i.e., Listeria monocytogenes) within the sample stream is assessed through bacterial culture techniques. Optimized pumping protocols can remove up to 50 % of debris from ground beef samples while percentage for preserved microorganisms can account for 95 % in relatively clean samples. However, comparison between inoculated turbid and clean samples (i.e., with and without ground beef debris) indicate some degree of interference between debris inertial lateral migration and hydrodynamic focusing of small microorganisms. Although this interference can lead to significant decrease in chip performance through loss of target bacteria, it remains possible to reach 70 % for sample recovery and more than 50 % for debris removal even in the most turbid samples tested. Due to the relatively simple design, the robustness of the inertial migration effect itself, the high operational flow rates and fabrication methods that leverage low-cost materials, the proposed device can have an impact on a wide range of applications where high-throughput separation of particles and biological species is of interest. [ABSTRACT FROM AUTHOR]

In this paper, we address the hydraulic flow of fluids on and within nanofabricated hydrophilic (as opposed to hydrophobic) surfaces, and the influence of an extended precursor film on the wetting dynamics of water penetrating a nano-textured array of posts that has no sealing surface (open-top). The width of the precursor film in front of the wetting edge δ is extraordinarily large, approximately 2mm for our device, and it drives the wetting penetration film in the open-top array. We discuss the way this thin precursor film controls the movement of the macroscopic wetting front Lw and its dynamics in the nanofabricated array of posts. Rapid and controllable transport of nanoparticles in such an open top array of posts, as controlled by the precursor film, is demonstrated using a bump array textured morphology. [ABSTRACT FROM AUTHOR]

We show that it is possible to direct particles entrained in a fluid along trajectories much like rays of light in classical optics. A microstructured, asymmetric post array forms the core hydrodynamic element and is used as a building block to construct microfluidic metamaterials and to demonstrate refractive, focusing, and dispersive pathways for flowing beads and cells. The core element is based on the concept of deterministic lateral displacement where particles choose different paths through the asymmetric array based on their size: !articles larger than a critical size are displaced laterally at each row by a post and move along the asymmetric axis at an angle to the flow, while smaller particles move along streamline paths. We create compound elements with complex particle handling modes by tiling this core element using multiple transformation operations; we show that particle trajectories can be bent at an interface between two elements and that particles can be focused into hydrodynamic jets by using a single inlet port. Although particles propagate through these elements in a way that strongly resembles light rays propagating through optical elements, there are unique differences in the paths of our particles as compared with photons. The unusual aspects of these modular, microfluidic metamaterials form a rich design toolkit for mixing, separating, and analyzing cells and functional beads On-chip. [ABSTRACT FROM AUTHOR]

Capsule: Between 1 992 and 2003 persecution appeared to be the main influential factor. Aims: To utilize temporal changes in the distribution and occupation of Golden Eagle territories in Scotland between the 1 992 and 2003 national censuses to assess potential causes of regional and national population trends, by examining spatial associations with a number of potential constraints on the population. Methods: The distribution of occupied and vacant territories in the 1992 and 2003 censuses were entered as layers in a Geographical Information System (GIS), along with boundaries of biogeographical regions (Natural Heritage Zones) for regional analyses. Additional GIS layers were created for potential factors that may constrain the eagle population: the distribution and abundance of persecution incidents, new commercial conifer forests, popular mountains for hillwalkers (as surrogates for recreational activity), and the density of sheep and Red Deer (as surrogates for carrion abundance), drawn from comparable time periods to the national eagle censuses. Analyses then looked for spatial associations between eagle territory status and those constraint factors that may have influenced change in territory status. Results: We found little evidence to suggest that recreational disturbance was influential on the occupation of Golden Eagle territories, although some local effects may have occurred and further analyses are warranted. We could find evidence of only a limited number of territories having being abandoned recently due to the planting of commercial conifer forests. We also rejected the hypothesis that changes in territory occupation between national Golden Eagle censuses were influenced by change in carrion abundance. By contrast, results were consistent with the hypothesis that persecution was influential in the observed change in territory occupation between censuses, so that occupied eagle territories tended to decline where persecution was probably still influential and tended to increase where persecution had probably declined. Conclusion: In accordance with earlier predictions based on models of the demographic influence of persecution, in the central and eastern Highlands where grouse moor management predominates, the eagle population continued to decline to levels where increasingly large areas of suitable habitat are unoccupied by breeding pairs. [ABSTRACT FROM AUTHOR]

We show the fractionation of whole blood components and isolation of blood plasma with no dilution by using a continuous-flow deterministic array that separates blood components by their hydrodynamic size, independent of their mass. We use the technology we developed of deterministic arrays which separate white blood cells, red blood cells, and platelets from blood plasma at flow velocities of 1,000 μm/sec and volume rates up to 1 μl/min. We verified by flow cytometry that an array using focused injection removed 100% of the lymphocytes and monocytes from the main red blood cell and platelet stream. Using a second design, we demonstrated the separation of blood plasma from the blood cells (white, red, and platelets) with virtually no dilution of the plasma and no cellular contamination of the plasma. [ABSTRACT FROM AUTHOR]

This article offers an introduction to service learning and a brief review of the research on the effects of service learning on academic and values development. It outlines in detail the history of Campus Compact, an organization of 517 college and university presidents founded in 1985, and its Project on Integrating Service with Academic Study. Lessons learned about institutionalizing service learning and information about resources for doing so are also summarized. The findings are based on a three-year, national project supported by the Ford Foundation and an anonymous donor, and two service-learning case studies. [ABSTRACT FROM AUTHOR]

In this paper, we present a simple, versatile method that creates patterns for cell migration studies using thermoplastic elastomer (TPE). The TPE material used here can be robustly, but reversibly, bonded to a variety of plastic substrates, allowing patterning of cultured cells in a microenvironment. We first examine the bonding strength of TPE to glass and polystyrene substrates and com-pare it to thermoset silicone-based PDMS under various conditions and demonstrate that the TPE can be strongly and reversibly bonded on commercially available polystyrene culture plates. In cell migration studies, cell patterns are templated around TPE features cored from a thin TPE film. We show that the significance of fibroblast cell growth with fetal bovine serum (FBS)-cell culture media compared to the cells cultured without FBS, analyzed over two days of cell culture. This simple approach allows us to generate cell patterns without harsh manipulations like scratch assays and to avoid damaging the cells. We also confirm that the TPE material is non-toxic to cell growth and supports a high viability of fibroblasts and breast cancer cells. We anticipate this TPE-based patterning approach can be further utilized for many other cell patterning applications such as in cell-to-cell communication studies. [ABSTRACT FROM AUTHOR]

The authors present a technique for the replication of molds for nanoimprint lithography (NIL) without solvents or etching. A thin hard amorphous silicon film is deposited onto imprinted or self-assembled polymer nanostructures by room-temperature conformal plasma-enhanced chemical vapor deposition. After attachment to another substrate and separation from the polymer original, the thin hard film forms a NIL mold that is the inverse of the polymer original. Using this technology, the authors demonstrate the replication of a 200 nm pitch grating mold and sub-50-nm features over wafer-scale areas without introducing additional line edge roughness associated with conventional replication methods. [ABSTRACT FROM AUTHOR]