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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.

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).

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.

DNA hybridization phenomena occurring on solid supports are not understood as clearly as aqueous phase hybridizations and mathematical models cannot predict some empirically obtained results. Ongoing research has identified important parameters but remains incomplete to accurately account for all interactions. It has previously been shown that the length of the overhanging (dangling) end of the target DNA strand following hybridization to the capture probe is correlated to interactions with the complementary strand in solution which can result in unbinding of the target and its release from the surface. We have developed an instrument for real-time monitoring of DNA hybridization on spherical particles functionalized with oligonucleotide capture probes and arranged in the form of a tightly packed monolayer bead bed inside a microfluidic cartridge. The instrument is equipped with a pneumatic module to mediate displacement of fluid on the cartridge. We compared this system to both conventional (passive) and centrifugally-driven (active) microfluidic microarray hybridization on glass slides to establish performance levels for the detection of single nucleotide polymorphisms. The system was also used to study the effect of the dangling end's length in real-time when the immobilized target DNA is exposed to the complementary strand in solution. Our findings indicate that increasing the length of the dangling end leads to desorption of target amplicons from bead-bound capture probes at a rate approaching that of the initial hybridization process. Finally, bead bed hybridization was performed with Streptococcus agalactiae cfb gene amplicons obtained from randomized clinical samples, which allowed for identification of group B streptococci within 5–15 min. The methodology presented here is useful for investigating competitive hybridization mechanisms on solid supports and to rapidly validate the suitability of microarray capture probes.

We present new observations of aqueous two-phase system (ATPS) thermodynamic and interfacial phenomena that occur inside sessile droplets due to water evaporation. Sessile droplets that contain polymeric solutions, which are initially in equilibrium in a single phase, are observed at their three-phase liquid-solid-air contact line. As evaporation of a sessile droplet proceeds, we find that submicron secondary water-in-water (W/W) droplets emerge spontaneously at the edges of the mother sessile droplet due to the resulting phase separation from water evaporation. To understand this phenomenon, we first study the secondary W/W droplet formation process on different substrate materials, namely, glass, polycarbonate (PC), thermoplastic elastomer (TPE), poly(dimethylsiloxane)-coated glass slide (PDMS substrate), and Teflon-coated glass slide (Teflon substrate), and show that secondary W/W droplet formation arises only in lower-contact-angle substrates near the three-phase contact line. Next, we characterize the size of the emergent secondary W/W droplets as a function of time. We observe that W/W drops are formed, coalesced, aligned, and trapped along the contact line of the mother droplet. We demonstrate that this W/W multiple emulsion system can be used to encapsulate magnetic particles and blood cells, and achieve size-based separation. Finally, we show the applicability of this system for protein sensing. This is the first experimental observation of evaporation-induced secondary W/W droplet generation in a sessile droplet. We anticipate that the phenomena described here may be applicable to some biological assay applications, for example, biomarker detection, protein sensing, and point-of-care diagnostic testing.

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.

We investigate the formation of suspended magnetic nanoparticle (MNP) assemblies (M-clouds) and their use for

A method modifying a vacuum-assisted UV micro-molding (VAUM) process is proposed for the fabrication of polymer two-level submicron high porosity membranes (2LHPM). The modified process allows for the fabrication of robust, large-area membranes over 5 × 5 cm2 with a hierarchical architecture made from a 200 nm-thick layer having submicron level pores (as small as 500 nm) supported by a 20 μm-thick layer forming a microporous structure with 10-15 μm diameter pores. The fabricated freestanding membranes are flexible and mechanically robust enough for post manipulation and filtration of cell samples. Very high white blood cell (WBC) capture efficiencies (≈97%) from healthy blood samples after red blood cell (RBC) lysis are demonstrated using a 3D-printed filter cartridge incorporated within these 2LHPM. A high release efficiency of ≈95% is also proved using the same setup. Finally, on-filter multistep immunostaining of captured cells is also shown.

The development of technology for the rapid, automated identification of bacterial culture isolates can help regulatory agencies to shorten response times in food safety surveillance, compliance, and enforcement as well as outbreak investigations. While molecular methods such as polymerase chain reaction (PCR) enable the identification of microbial organisms with high sensitivity and specificity, they generally rely on sophisticated instrumentation and elaborate workflows for sample preparation with an undesirably high level of hands-on engagement. Herein, we describe the design, operation and performance of a lab-on-a-chip system integrating thermal lysis, PCR amplification and microarray hybridization on the same cartridge. The assay is performed on a centrifugal microfluidic platform that allows for pneumatic actuation of liquids during rotation, making it possible to perform all fluidic operations in a fully-automated fashion without the need for integrating active control elements on the microfluidic cartridge. The cartridge, which is fabricated from hard and soft thermoplastic polymers, is compatible with high-volume manufacturing (e.g., injection molding). Chip design and thermal interface were both optimized to ensure efficient heat transfer and allow for fast thermal cycling during the PCR process. The integrated workflow comprises 14 steps and takes less than 2 h to complete. The only manual steps are related to loading of the sample and reagents on the cartridge as well as fluorescence imaging of the microarray. On-chip lysis and PCR amplification both provided results comparable to those obtained by bench-top instrumentation. The microarray, incorporating a panel of oligonucleotide probes for multiplexed detection of seven enterohemorrhagic E. coli priority serotypes, was implemented on a cyclic olefin copolymer substrate using a novel activation scheme that involves the conversion of hydroxyl groups (derived from oxygen plasma treatment) into reactive cyanate ester using cyanogen bromide. On-chip hybridization was demonstrated in a non-quantitative fashion using fluorescently-labelled gene markers for E. coli O157:H7 (rfbO157, eae, vt1, and vt2) obtained through a multiplexed PCR amplification step.

The development of technology for the rapid, automated identification of bacterial culture isolates can help regulatory agencies to shorten response times in food safety surveillance, compliance, and enforcement as well as outbreak investigations. While molecular methods such as polymerase chain reaction (PCR) enable the identification of microbial organisms with high sensitivity and specificity, they generally rely on sophisticated instrumentation and elaborate workflows for sample preparation with an undesirably high level of hands-on engagement. Herein, we describe the design, operation and performance of a lab-on-a-chip system integrating thermal lysis, PCR amplification and microarray hybridization on the same cartridge. The assay is performed on a centrifugal microfluidic platform that allows for pneumatic actuation of liquids during rotation, making it possible to perform all fluidic operations in a fully-automated fashion without the need for integrating active control elements on the microfluidic cartridge. The cartridge, which is fabricated from hard and soft thermoplastic polymers, is compatible with high-volume manufacturing (e.g., injection molding). Chip design and thermal interface were both optimized to ensure efficient heat transfer and allow for fast thermal cycling during the PCR process. The integrated workflow comprises 14 steps and takes less than 2 h to complete. The only manual steps are related to loading of the sample and reagents on the cartridge as well as fluorescence imaging of the microarray. On-chip lysis and PCR amplification both provided results comparable to those obtained by bench-top instrumentation. The microarray, incorporating a panel of oligonucleotide probes for multiplexed detection of seven enterohemorrhagic E. coli priority serotypes, was implemented on a cyclic olefin copolymer substrate using a novel activation scheme that involves the conversion of hydroxyl groups (derived from oxygen plasma treatment) into reactive cyanate ester using cyanogen bromide. On-chip hybridization was demonstrated in a non-quantitative fashion using fluorescently-labelled gene markers for E. coli O157:H7 (rfbO157, eae, vt1, and vt2) obtained through a multiplexed PCR amplification step.

We employed variable temperature chemical force microscopy (VT-CFM) using tips silanized with four different hydro- and hydrofluoroalkyl self-assembling monolayers (SAMs) interacting with a thin-film of poly(cyclic olefin), (PCO) to model the hot-embossing stamp-polymer interaction over a temperature range spanning the glass transition of the PCO.

A versatile method for fabricating both plasmonic and conductive metal structures in situ in polymer materials is presented. The process relies on UV induced radical polymerization of acrylic resin and gold nanoparticle formation by the reduction of a gold precursor in the presence of a photoinitiator. The room temperature fabrication process provides control over nanoparticle size, growth and distribution within the polymer matrix., 2020 IEEE 20th International Conference on Nanotechnology (IEEE-NANO), July 29-31, 2020, Montreal, QC, Canada

Hierarchical micro-nanostructures comprising nanopillars superimposed onto micropillars were successfully fabricated over larger areas on thermoplastic cyclic olefin polymer (COP) substrates. Both the nanostructures (100 nm to 500 nm diameter) and microstructures (14-20 μm diameter) were hot-embossed monolithically using an intermediate fluorinated polymer mold replicated from a Si master. The as-fabricated thermoplastic hierarchical micro-nanopillar surfaces are superhydrophobic with advancing contact angles greater than 150°. Superhydrophobic surfaces with static contact angle as large as 167° were achieved on COP substrates using nanopillars as small as 150 nm and following post-fabrication fluorocarbon coating. The sliding angle of a water drop on the superhydrophobic surface is 2.5°., 2020 IEEE 20th International Conference on Nanotechnology (IEEE-NANO), July 29-31, 2020, Montreal, QC, Canada

A multiplexed droplet PCR (mdPCR) workflow and detailed protocol for determining epigenetic-based white blood cell (WBC) differential count is described, along with a thermoplastic elastomer (TPE) microfluidic droplet generation device. Epigenetic markers are used for WBC subtyping which is of important prognostic value in different diseases. This is achieved through the quantification of DNA methylation patterns of specific CG-rich regions in the genome (CpG loci). In this paper, bisulfite-treated DNA from peripheral blood mononuclear cells (PBMCs) is encapsulated in droplets with mdPCR reagents including primers and hydrolysis fluorescent probes specific for CpG loci that correlate with WBC sub-populations. The multiplex approach allows for the interrogation of many CpG loci without the need for separate mdPCR reactions, enabling more accurate parametric determination of WBC sub-populations using epigenetic analysis of methylation sites. This precise quantification can be extended to different applications and highlights the benefits for clinical diagnosis and subsequent prognosis.

We present here a new method for controlling the droplet size in step emulsification processes on a centrifugal microfluidic platform, which, in addition to the centrifugal force, uses pneumatic actuation for fluid displacement. We highlight the importance of the interplay between buoyancy effects and the flow rate at the step junction, and provide a simple analytical model relating these two quantities to the size of the droplets. Numerical models as well as experiments with water-in-oil emulsions are performed in support of the proposed model.

This paper describes the development of an on-chip nucleic acid (NA) extraction assay from whole blood using a centrifugal microfluidic platform that allows for pneumatic actuation of liquids during rotation. The combination of pneumatic and centrifugal forces makes it possible to perform fluidic operations without the need for integrating active control elements on the microfluidic cartridge. The cartridge is fabricated from thermoplastic polymers (e.g., Zeonor 1060R) and features a simple design that is compatible with injection molding. In addition, the cartridge is interfaced with two external vials for off-chip storage of the blood sample and retrieval of the eluted NA solution, respectively. On-chip capture of NAs is performed using an embedded solid-phase extraction matrix composed of commercial glass microfiber filters (Whatman GF/D and GF/F). The yield of the automated, on-chip extraction protocol, determined by measuring absorbance at 260 nm, is comparable to some of the best manually operated kits (e.g., Qiagen QIAamp DNA Mini Kit) while providing low assay-to-assay variability due to the high level of control provided by the platform for each processing step. The A260/A280 and A260/A230 ratios of the absorbance spectra also reveal that protein contamination of the sample is negligible. The capability of the pneumatic platform to circulate air flux through the microfluidic conduit was used to dry leftover ethanol residues retained in the capture matrix during washing. This method, applied in combination with localized heating, proved effective for reducing ethanol contamination in eluted samples from ∼12% to 1% (v/v).

Transformative advances in metagenomics are providing an unprecedented ability to characterize the enormous diversity of microorganisms and invertebrates sustaining soil health and water quality. These advances are enabling a better recognition of the ecological linkages between soil and water, and the biodiversity exchanges between these two reservoirs. They are also providing new perspectives for understanding microorganisms and invertebrates as part of interacting communities (i.e. microbiomes and zoobiomes), and considering plants, animals, and humans as holobionts comprised of their own cells as well as diverse microorganisms and invertebrates often acquired from soil and water. The Government of Canada's Genomics Research and Development Initiative (GRDI) launched the Ecobiomics Project to coordinate metagenomics capacity building across federal departments, and to apply metagenomics to better characterize microbial and invertebrate biodiversity for advancing environmental assessment, monitoring, and remediation activities. The Project has adopted standard methods for soil, water, and invertebrate sampling, collection and provenance of metadata, and nucleic acid extraction. High-throughput sequencing is located at a centralized sequencing facility. A centralized Bioinformatics Platform was established to enable a novel government-wide approach to harmonize metagenomics data collection, storage and bioinformatics analyses. Sixteen research projects were initiated under Soil Microbiome, Aquatic Microbiome, and Invertebrate Zoobiome Themes. Genomic observatories were established at long-term environmental monitoring sites for providing more comprehensive biodiversity reference points to assess environmental change.

This paper reports the characteristics of thermoplastic elastomer based normally closed microvalves with microstructured valve seats. Compared to the state of the art, we demonstrate, for the first time, a solution to the problematic issue of valve bonding that is frequently encountered with microfluidic valves during device assembly. Our configuration enables industrial manufacturing of fully thermoplastic microfluidic devices integrated with microvalves for a wide variety of biomedical applications., 2020 IEEE 33rd International Conference on Micro Electro Mechanical Systems (MEMS), January 18-22, 2020, Vancouver, BC, Canada

Health maintenance and disease prevention in astronauts are of high priority during space missions. To achieve this aim, basic science of molecular biology has to be joined with modern noninvasive tools allowing high-fidelity research on diagnosing and monitoring of the astronauts’ health status. Experts from biology, medicine, and engineering should merge their expertise to enable additive effects for development and improvement of stress and immune-monitoring tools under extreme environmental conditions. This will hereby ensure not only the success of space missions, especially when leaving the Earth orbit in the future, but also will provide important impetus for many other applications on Earth as well.

Epigenetic markers attract increasing attention for the study of phenotypic variations, which has led to the investigation of cell-lineage DNA methylation patterns that correlate with human leukocyte populations for obtaining counts of white blood cell (WBC) subsets. Current methods of DNA methylation analysis involve genome sequencing or loci-specific quantitative PCR (qPCR). Herein, a multiplexed digital droplet PCR (ddPCR) workflow for determining epigenetic-based WBC differential count is described for the first time. A microfluidic emulsification device fabricated from a commercially available thermoplastic elastomer (e.g., Mediprene) promotes customizability and cost-effectiveness of the methodology, which are prerequisites for translation into clinical and point-of-care diagnostics. Bisulfite-treated DNA from peripheral blood mononuclear cells and whole blood is encapsulated in droplets with ddPCR reagents containing primers and fluorescent hydrolysis probes specific for CpG loci correlated with WBC sub-population types. The method enables multiplexed detection of various methylation sites within a single droplet. Both qPCR and immunofluorescence staining (IF) were conducted to validate the capacity of the ddPCR methodology to accurately determine WBC sub-populations using epigenetic analysis of methylation sites. ddPCR results correlated closely to cell proportions obtained using IF, whereas qPCR significantly underestimated these values for both high and low copy number gene targets.

A stretchable capacitive strain sensor based on thermoplastic elastomer (TPE) microfluidics embedded with liquid metal was successfully fabricated. Benefiting from the unique self-bonding feature of the TPE materials, the capacitance of a sensor was enhanced by increasing the number of TPE layers without changing the footprint of the sensor. The change of the sensor capacitance is linearly dependent on the strain with small hysteresis. The gauge factor of the sensor is closed to the theoretical maximum value 1. The capacitance of the sensor is very stable and repeatable after stretching and releasing for 5000 cycles., 2019 IEEE SENSORS, October 27-30, 2019, Montreal, QC, Canada

SummaryCirculating tumor cells (CTCs) are rare (few cells per milliliter of blood) and mostly isolated as single cell CTCs (scCTCs). CTC clusters (cCTCs), even rarer, are of growing interest, notably because of their higher metastatic potential, but very difficult to isolate. Here, we introduce gravity-based microfiltration (GµF) for facile isolation of cCTCs. We identify cluster break-up as a confounding cause, and achieve ∼85% capture efficiency. GµF from orthotopic ovarian cancer mouse models and from 10 epithelial ovarian cancer (EOC) patients uncovered cCTCs in every case, with between 2-100+ cells. cCTCs represented between 5-30% of all CTC events, and 10-80% of captured CTCs were clustered; remarkably, in two patients, more CTCs were circulating as cCTCs than scCTCs. GµF uncovered the unexpected prevalence, frequency and sometimes large size of cCTCs in EOC patients with either metastatic and localized disease, and motivates additional studies to uncover their properties and role in disease progression.

This review is an account of centrifugal microfluidic systems that use various actuation strategies in addition to intrinsic centrifugal forces to accurately regulate the motion of fluids during rotation. Platforms that integrate active methods of pumping and flow control render centrifugal microfluidics more versatile as they facilitate integration and process automation by enabling (or improving the reliability of) important fluidic functions, such as metering, aliquoting, valving, flow switching, mixing, and inward pumping. Principles and working mechanisms underlying these strategies are described in the context of recent trends in instrument design and development where centrifugal platforms have been equipped with pneumatic, magnetic or electromechanical actuators serving as pumping and valving elements. The potential of these platforms to perform complex bioanalytical assays in an automated fashion is illustrated by several examples, which include on-chip preparation of aliquot libraries, nucleic acid purification, amplification and analysis as well as blood separation.

Circulating tumor cells (CTCs) have been linked to cancer progression but are difficult to isolate, as they are very rare and heterogeneous, covering a range of sizes and expressing different molecular receptors. Filtration has emerged as a simple and powerful method to enrich CTCs but only captures cells above a certain size regardless of molecular characteristics. Here, we introduce antibody-functionalized microfilters to isolate CTCs based on both size and surface receptor expression. We present a 3D printed filtration cartridge with microfabricated polymer filters with 8, 10, 12, 15, or 20 μm-diameter pores. Pristine filters were used to optimize sample dilution, rinsing protocol, flow rate, and pore size, leading to >80% for the recovery of spiked cancer cells with very low white blood cell contamination (96%. These results suggest that filtration can be optimized to target specific CTC characteristics such as size and receptor expression and that a diverse range of CTCs may be captured using particular combinations of pore size, filtration parameters, and antibody functionalization.

In the clinical and pharmacological fields, there is a need for the production of tissue-engineered small-diameter blood vessels. We have demonstrated previously that the extracellular matrix (ECM) produced by fibroblasts can be used as a scaffold to support three-dimensional (3D) growth of another cell type. Thus, a resistant tissue-engineered vascular media can be produced when such scaffolds are used to culture smooth muscle cells (SMCs). The present study was designed to develop an anisotropic fibroblastic ECM sheet that could replicate the physiological architecture of blood vessels after being assembled into a small diameter vascular conduit. Anisotropic ECM scaffolds were produced using human dermal fibroblasts, grown on a microfabricated substrate with a specific topography, which led to cell alignment and unidirectional ECM assembly. Following their devitalization, the scaffolds were seeded with SMCs. These cells elongated and migrated in a single direction, following a specific angle relative to the direction of the aligned fibroblastic ECM. Their resultant ECM stained for collagen I and III and elastin, and the cells expressed SMC differentiation markers. Seven days after SMCs seeding, the sheets were rolled around a mandrel to form a tissue-engineered vascular media. The resulting anisotropic ECM and cell alignment induced an increase in the mechanical strength and vascular reactivity in the circumferential direction as compared to unaligned constructs. Copyright © 2016 John Wiley & Sons, Ltd.

We describe the translation of a cloth-based hybridization array system (CHAS), a colorimetric DNA detection method that is used by food inspection laboratories for colony screening of pathogenic agents, onto a microfluidic chip format. We also introduce an articulated centrifugal platform with a novel fluid manipulation concept based on changes in the orientation of the chip with respect to the centrifugal force field to time the passage of multiple components required for the process. The platform features two movable and motorized carriers that can be reoriented on demand between 0 and 360° during stage rotation. Articulation of the chip can be used to trigger on-the-fly fluid dispensing through independently addressable siphon structures or to relocate solutions against the centrifugal force field, making them newly accessible for downstream transfer. With the microfluidic CHAS, we achieved significant reduction in the size of the cloth substrate as well as the volume of reagents and wash solutions. Both the chip design and the operational protocol were optimized to perform the entire process in a reliable, fully automated fashion. A demonstration with PCR-amplified genomic DNA confirms on-chip detection and identification of Escherichia coli O157:H7 from colony isolates in a colorimetric multiplex assay using rfbO157, fliCH7, vt1, and vt2 genes.

Pathogenic bacteria can be a major cause of illness from environmental sources as well as the consumption of contaminated products, giving rise to public health concerns globally. The surveillance of such living organisms in food and water supplies remains an important challenge in mitigating their deleterious societal effects. Here, we have developed an optimized bioorthogonal non-canonical amino acid tagging approach to the imaging, capture, and interrogation of shigatoxigenic/verotoxigenic Escherichia coli (VTEC) and Listeria that enables the distinction between living wild-type pathogenic bacteria. The approaches utilize homopropargylglycine (HPG), as well as optimized growth media, that restricts endogenous methionine biosynthesis in a variety of species of public health concern. Endogenous methionine residues are then replaced with HPG, which can then be modified using a myriad of compatible bioorthogonal reactions for tagging of exclusively live bacteria. The methods reported allow for the very rapid screening and identification of living pathogenic organisms.

We present an integrated microfluidic system for performing isolation and concentration of Phytophthora ramorum pathogens using a chip whose working principle is based on inertial lateral migration in curving flows. The chip was fabricated from multiple layers of thermoplastic polymers and features an embedded spiral separation channel along with peristaltic microvalves for fluidic operation and process control. A pumping system paired with a fully programmable pressure manifold is used to boost concentration levels by recirculating the sample liquid multiple times through the separation chip, making it possible to reduce sample volumes from 10 to 1 mL or less. The system was calibrated using fluorescent polymer particles with a nominal diameter of 30 µm which is comparable to that of P. ramorum sporangia. The separation process has been shown to be highly effective and more than 99% of the beads can be recovered in the concentrated batch. Experiments conducted with P. ramorum sporangia have shown that a 5.3-fold increase in pathogen content with 95% recovery can be achieved using three subsequent concentration cycles. The utility of the method has been validated by processing a sample derived from infested Rhododendron leaves where a 6.1-fold increase in the concentration of P. ramorum has been obtained after four concentration cycles. Although specifically designed and demonstrated for sporangia of P. ramorum, the method and related design rules can easily be extended to other microbial organisms, effectively supporting bioanalytical applications where efficient, high-throughput separation of target species is of primary concern.

Legionellosis has been and continues to be a life-threatening disease worldwide, even in developed countries. Given the severity and unpredictability of Legionellosis outbreaks, developing a rapid, highly specific, and sensitive detection method is thus of great pertinence. In this paper, we demonstrate that sub-femtomole levels of 16s rRNA from pathogenic Legionella pneumophila can be timely and effectively detected using an appropriate designed capture, detector probes, and a QD SPRi signal amplification strategy. To achieve specific and sensitive detection, optimal hybridization conditions and parameters were implemented. Among these parameters, fragmentation of the 16s rRNA and further signal amplification by QDs were found to be the main parameters contributing to signal enhancement. The appropriate design of the detector probes also increased the sensitivity of the detection system, mainly due to secondary structure of 16s rRNA. The use of 16s rRNA from L. pneumophila allowed for the detection of metabolically active pathogens with high sensitivity. Detection of 16s rRNA in solutions as diluted as 1. pM at 450. μL (0.45. femtomole) was achieved in less than 3. h, making our approach suitable for the direct, timely, and effective detection of L. pneumophila within man-made water systems. © 2013 Elsevier B.V.

There is a lack of an optimal transplant material for small calibre blood vessels. This could be overcome by tissue engineering. The optimal construct is to be derived from autologous cells and present mechanical resistance comparable to the gold standard, autologous vessels such as the internal mammary artery or the saphenous vein. Our laboratory has developed the self-assembly approach to produce tissue sheets that can be rolled into such vessel substitutes. Over the years, many improvements have been made to the technique to facilitate smooth muscle cell culture and to produce vascular media substitutes with higher circumferential mechanical resistance.

Numerous samples of magnetite@silica and magnetite@silica@silane core-shell nanoparticles, previously used as prodrugs, were prepared by an experienced chemist, using the same identical equipment and the same lots of reagents. Their surface analyses showed batch-to-batch chemical variations: no two batches were found to have the same surface chemistries, showing unexpected Si-O bond scission and amine oxidation. Because the preparations used reactions recognized to be mild, and bond scission and oxidation were never previously reported for similar reactions on larger surfaces, the Fe3O4 nanoparticles that form the nanoparticle core appear to have acted as catalysts for these side reactions. The intended use of these nanoparticles, as drug carriers, is discussed in terms of cytotoxicological and biomedical consequences. © 2012 Elsevier Inc.

Superparamagnetic core/shell nanoparticles, composed of a free-standing Fe3O4 magnetic core inside a hollow nanoporous silica shell, have been synthesized, and their structures and magnetic and drug release behavior were systematically investigated. The hollow/nanoporous silica shells, containing secondary amine groups, were functionalized with 1, 2-cyclohexanedicarboxylic anhydride as click linkers, producing pH-sensitive amides and terminal carboxylic groups for the conjugation and release of the anticancer drug doxorubicin. The results show that the effective release occurred at pH 5 and was 3 times higher than that at pH 7.4. The kinetics of release was assumed as a Fickian diffusion process and fitted by the Higuchi model to uncover an intrinsic relationship of the released amount with time, temperature, and pH. © 2010 American Chemical Society.

Core/shell Fe3O4−Au/silica nanoparticles with controllable fluorescein doped into nanoporous silica shells are synthesized. The quantum yield of fluorescein significantly increases through a synthetic electromagnetic field coupling of the dumbbell-like Fe3O4−Au cores. Theoretical simulation further proves that the anomalously plasmonic oscillation of Fe3O4−Au nanoparticles is a dominant contributor to the fluorescence enhancement.

We use a three-dimensional multiphase lattice-Boltzmann model to study basic operations such as transport, merging and splitting of nanoliter water droplets actuated by electrowetting in digital microfluidic devices. In a first step, numerical and analytical predictions for the droplet transport velocity are compared and very good agreement is obtained for a wide range of contact angles. The same algorithm is employed then to study the dynamics of the splitting processes at different contact angles and different geometries of the cell. The configuration of the liquid droplet involved in a splitting process and the dependence of the splitting time on the transport velocity are also investigated and phenomenological laws describing these processes are also proposed.

Background The presence of circulating tumor cells (CTCs) in blood is correlated with disease progression in many cancers. Their prognosis value in ovarian cancer is still under debate.1 CTCs are heterogeneous in size and marker expression, and sub-populations with various metastatic potentials have been identified. CTC clusters, although rare and difficult to isolate, have emerged as a possible driver of metastasis owing to ~50-time higher metastatic potential than single CTCs.2 Only few methods have emerged to capture clusters, and are often complex and cumbersome, limiting our understanding of the role of clusters in metastasis. Here, we present a new filtration method for the selective capture of CTC clusters from blood and found clusters in 12/12 epithelial ovarian cancer (EOC) patients. Method Cluster capture was performed by filtration using a 3D printed cartridge3 and filters4 with pore diameters of 8, 10, 12, 15, 20 or 28 μm. We developed a gravity-driven process, generating reduced shear stress, and optimized capture using blood (1:6, v/v, in PBS) spiked with OV-90 and OVCAR-3 ovarian cancer single cells and clusters. Blood samples from 12 EOC patients were filtered. Clusters can be stained and imaged on the filter, or released for downstream analysis. Results Using the gravity-setup, we were able to selectively capture clusters with good integrity and with a rate that outperforms other technologies to the best of our knowledge. Viable CTC clusters, with 2 to >100 cells, were captured from 12/12 EOC patients. Their size distribution was surprisingly similar between patients. Small clusters (2-3 cells) were the most frequent, and this frequency decreased as their size increased. The molecular characterization of the captured clusters revealed a low and localized, heterogeneous expression of EpCAM (epithelial cell adhesion molecule), in combination with a widespread expression of c-MET (hepatocyte growth factor receptor) in all patients, suggesting a mesenchymal-like profile. Conclusion Using the gravity-filtration setup, CTC clusters were captured from the blood of all patients tested, suggesting that clusters are much more widespread than anticipated, and are in fact the norm rather than the exception. The cluster size distribution was conserved between patients with small clusters dominating, and some rare, very large clusters. Cluster staining revealed a mesenchymal profile, in agreement with a higher metastatic potential. Together, these results suggest that clusters should significantly contribute to disease progression, a hypothesis, which may be explored using our facile and selective method. References 1. Y. Zhou, et al. PLoS ONE 2015, 10, e0130873. 2. N. Aceto, et al. Cell 2014, 158, 1110. 3. A. Meunier, et al. Anal. Chem. 2016, 88, 8510. 4. J. A. Hernandez-Castro, et al. LOC 2017, 17, 1960 Citation Format: Anne Meunier, Sara Kheireddine, J. Alejandro Hernández-Castro, Benjamin Péant, Diane Provencher, Anne-Marie Mes-Masson, Teodor Veres, David Juncker. Clusters of circulating tumor cells were selectively isolated in the blood of 12/12 epithelial ovarian cancer patients using facile gravity-flow-based filtration method adapted to clinical use [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1562.

We present the synthesis and magnetic properties of high quality uncoated and gold-coated iron oxide magnetic nanoparticles. The structural properties of these nanoparticles are investigated by transmission electron microscopy, UV–visible spectroscopy and X-ray diffraction. Experimental results and theoretical simulations indicate that the synthesized nanoparticles present a very good monodispersity, and well defined size and shape. The coercive field of these particles is identified by low-temperature first-order reversal curves and the results used in order to fit zero-field-cooled magnetization processes with theoretical models. The identification of the parameters in this analysis suggests that the coating process hardly affects the morphology and the overall magnetic properties of the cores inside coated particles.

This paper investigates the fabrication of substrates for surface-enhanced Raman scattering (SERS) using hard thermoplastic polymers in conjunction with sputtering and electroless deposition of a thin Au or Ag film. In contrast to silicon or glass, plastic materials promote the replication of nanoscale structures over large areas in a rapid and cost-effective manner using molding or embossing techniques. However, synthetic polymers are Raman-active compounds, providing a source of interference with signals arising from reporter molecules that may be used for SERS detection. Moreover, most plastics melt and undergo degradation when exposed to a focused laser beam, resulting in irreversible loss of the SERS-active metal features in the detection area. We address these issues and show that proper decoration of the plastic surface with a metal overlayer can suppress Raman background signal of the polymer substrate, as well as any adverse effect arising from laser illumination.

This paper describes the patterning of DNA arrays on plastic surfaces using an elastomeric, two dimensional (2D) microcapillary system (μCS). Fluidic structures were realized through hot embossing lithography using Versaflex® CL30. Like elastomers based on poly(dimethylsiloxane) (PDMS), this thermoplastic block co-polymer is able to seal a surface in a reversible manner, making it possible to confine DNA probes with a level of control that is unparalleled using standard microspotting techniques. We focus on μCSs that support arrays comprising up to 48 spots each being 45 μm in diameter. Substrates were fabricated from two hard termoplastic materials - poly(methylmethacrylate) (PMMA) and a polycyclic olefin (e.g., Zeonor® 1060R) - which were both activated with N-hydroxysuccinimide (NHS) ester to mediate covalent attachment of DNA molecules. The approach was exemplified by using 0.25 to 32 μM solutions of amino-modified oligonucleotides labeled with either Cy3 or Cy5 fluorescent dye in phosphate buffered saline (PBS), allowing for a direct and sensitive characterization of the printed arrays. Solutions were incubated for durations of 1 to >48 h at22, 30 and 40℃ to probe the conditions for obtaining uniform spots of high fluorescence intensity. The length (l) and depth (d) of microfluidic supply channels were both important with respect to depletion as well as evaporation of the solvent. While selective activation of the substrate proved helpful to limit unproductive loss of oligonucleotides along trajectories, incubation of solution in a humid environment was necessary to prevent uncontrolled drying of liquid, keeping the immobilization process intact over extended periods of time. When combined, these strategies effectively promoted the formation of high-quality DNA arrays, making it possible to arrange multiple probes in parallel with a high degree of uniformity. Moreover, we show that resultant arrays are compatible with standard hybridization protocols, which allowed for reliable discrimination of individual stands when exposed to a specific ssDNA target molecule., available, unlimited, public

This work reports on a dynamically configurable micro-array surface plasmon resonance biochip platform. The platform comprises a digital electrowetting-on-dielectric (EWOD) microfluidic device tailored to surface plasmon resonance imaging (SPRi). We demonstrate its application for simultaneous immobilization of different DNA probes at the designated detection sites on-chip from sub-microL volume solutions in combination with multichannel label-free real-time detection of subsequent hybridization reactions. Successful on-chip DNA probe dilution and immobilization is also demonstrated using SPRi hybridization detection. Furthermore, active control of the immobilized probe density and orientation is achieved under an applied potential using the electric interface of the EWOD device. For low probe densities, under negative applied potential, the DNA hybridization efficiency is enhanced compared to passive probe immobilization, yielding a two-fold SPR signal increase within only 8min of hybridization. EWOD microfluidic platform coupled with SPRi promises to dramatically increase the speed of detection and quantification of biomolecular interactions while reducing reagent consumption. The proposed system would enable the development of high-throughput, rapid and ultrasensitive detection of biomolecules beyond DNA microarray applications.

In vivo, cardiomyocytes are exposed to multiple biochemical and physical cues including topographical and electrical cues. During prolonged in vitro cultivation in standard tissue culture set-ups, cardiomyocytes are known to de-differentiate due to the lack of appropriate micro-environmental cues. Most currently available cell culture systems provide only a single biophysical cue, thus development of advanced cell cultivation systems incorporating multiple cues is urgently needed. We report here the development of a microfabricated system, incorporating topographical and electrical cues on a single chip, which enables cultivation of differentiated cardiomyocytes. The cell culture chips were created by hot embossing of polystyrene, to create microgrooves and microridges of precisely defined depth, width and periodicity. Substrates consisting of 0.5 microm-wide grooves and 0.5 microm-wide ridges (1 microm period) and those consisting of 3 microm-wide grooves and 1 microm-wide ridges (4 microm period) were investigated, with smooth surfaces used as controls. The depth of the microgrooves was 400 nm. The two gold electrodes were electrodeposited 1 cm apart such that the microgrooves in-between were oriented either parallel or perpendicular to the electrodes, enabling studies of interaction between topographical and electrical cues. Neonatal rat cardiomyocytes cultivated on microgrooved substrates for 7 days were elongated and aligned along the microgrooves forming a well developed contractile apparatus, as evidenced by sarcomeric alpha-actinin staining, with a more pronounced effect on substrates with 1 microm compared to 4 microm periodicity. Importantly, simultaneous application of biphasic electrical pulses and topographical cues resulted in gap junctions confined to the cell-cell end junctions rather than the punctate distribution found in neonatal cells. Electrical field stimulation further enhanced cardiomyocyte elongation when microgrooves were oriented parallel to the electric field. Due to the compatibility of the described cell culture chips with fluorescence and optical microscopy as well as the ability to independently control field stimulation parameters, biochemical and topographical cues on each chip, this system may in the future become a useful tool in drug development and maturation of cardiomyocytes derived from stem cells.

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For both research and diagnostic purposes, the ability to detect low levels of proteins in a cost- and time-effective manner is essential. In this study, the cytokine TNF-alpha (tumor necrosis factor-alpha), a widely used protein indicator of inflammatory response, was chosen to demonstrate the ability of the dual-network microfluidic ELISA (enzyme-linked immunosorbent assay) platform developed by the authors to rapidly quantify low concentrations of this biomarker in serum. Through the optimization of several experimental parameters, the system was shown to meet the requirements for fundamental and applied studies, while also being relevant for challenging clinical applications such as the diagnosis of septic patients. A sensitivity of 45 pg/mL (2.6 pM) in both culture medium and serum, with inter- and intravariations of less than 15%, was attained for the quantification of human TNF-alpha to a concentration of up to 500 pg/mL. The overall time for completion of the assay in eight parallel reactions was less than 1 h.

Excellent quality superparamagnetic iron oxide coated iron nanoparticles were prepared via one pot synthesis with narrow size distribution. These particles were subsequently coated with FexOy, FePt or Pt shell to give long term protection to the highly reactive zero-valent iron core. Clear core-shell morphology was observed in the TEM images of FexOy and FePt coated particles and the magnetic properties suggested that these shells provided good protection to the core from oxidation, in particular iron oxide coated particles retained their magnetism for well over a year. FePt coated particles show superior magnetic properties to FePt particles alone and were found to be promising in biological applications. The magnetic studies further revealed that the saturation moments of the resultant particles were dependent on the type of shell used for encapsulation. Surface modifications of these particles were done with suitable hydrophilic ligands and they showed excellent stability and solubility in water and different buffer solutions which reveals the suitability of these nanoparticles for biomedical research.