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We demonstrate a highly efficient DLD separation device and process that is driven by a paper wick yet allows direct collection of products from reservoirs.

A viscoelastic microfluidic device for shape-based separation of drug-treated Escherichia coli.

SUMMARY Background Biofilm formation has been shown to be associated with damaged areas of endoscope channels. It was hypothesized that the passage of instruments and brushes through endoscope channels during procedures and cleaning contributes to channel damage, bacterial attachment and biofilm formation. Aim To compare surface roughness and bacterial attachment in used and new endoscope channels in vivo and in vitro. Methods Surface roughness of 10 clinically used (retired) and seven new colonoscope biopsy channels was analysed by a surface profiler. For the in-vitro study, a flexible endoscope biopsy forceps was passed repeatedly through a curved 3.0-mm-diameter Teflon tube 100, 200 and 500 times. Atomic force microscopy was used to determine the degree of inner surface damage. The number of Escherichia coli or Enterococcus faecium attached to the inner surface of the new Teflon tube and the tube with 500 forceps passes in 1 h at 37oC was determined by culture. Results The average surface roughness of the used biopsy channels was found to be 1.5 times greater than that of the new biopsy channels (P=0.03). Surface roughness of Teflon tubes with 100, 200 and 500 forceps passes was 1.05-, 1.12- and 3.2-fold (P=0.025) greater than the roughness of the new Teflon tubes, respectively. The number of E. coli and E. faecium attached to Teflon tubes with 500 forceps passes was 2.9-fold (P=0.021) and 4.3-fold (P=0.004) higher compared with the number of E. coli and E. faecium attached to the new Teflon tubes, respectively. Conclusion An association was found between endoscope usage with damage to the biopsy channel and increased bacterial attachment.

Sub-micrometer particles (0.10-1.0 μm) are of great significance to study, e.g., microvesicles and protein aggregates are targets for therapeutic intervention, and sub-micrometer fluorescent polystyrene (PS) particles are used as probes for diagnostic imaging. Focusing of sub-micrometer particles - precisely control over the position of sub-micrometer particles in a tightly focused stream - has a wide range of applications in the field of biology, chemistry and environment, by acting as a prerequisite step for downstream detection, manipulation and quantification. Microfluidic devices have been attracting great attention as desirable tools for sub-micrometer particle focusing, due to their small size, low reagent consumption, fast analysis and low cost. Recent advancements in fundamental knowledge and fabrication technologies have enabled microfluidic focusing of particles at sub-micrometer scale in a continuous, label-free and high-throughput manner. Microfluidic methods for the focusing of sub-micrometer particles can be classified into two main groups depending on whether an external field is applied: 1) passive methods, which utilize intrinsic fluidic properties without the need of external actuation, such as inertial, deterministic lateral displacement (DLD), viscoelastic and hydrophoretic focusing; and 2) active methods, where external fields are used, such as dielectrophoretic, thermophoretic, acoustophoretic and optical focusing. This article mainly reviews the studies on the focusing of sub-micrometer particles in microfluidic devices over the past 10 years. It aims to bridge the gap between the focusing of micrometer and nanometer scale (1.0-100 nm) particles and to improve the understanding of development progress, current advances and future prospects in microfluidic focusing techniques.

We are interested in the opto-mechanical manipulation of nanodiamonds with a high concentration of SiV centers. Optical tweezers usually rely on the interaction of light and the ‘bulk’ polarizability of the nanoparticle itself. Here we exploit the polarizability of electronic resonances of optical centers embedded in the solid-state matrix to enhance the optical forces. This effect becomes particularly relevant for ensembles of active centers. The emitters being closely packed in a sub-wavelength volume, can act cooperatively, enhancing further the optical forces. This opens the possibility to apply the powerful toolbox of atomic physics for the quantum manipulation of ‘massive’ mesoscopic objects.

Red blood cell (RBC) shape change under static and dynamic shear stress has been a source of interest for at least 50 years. High-speed time-lapse microscopy was used to observe the rate of deformation and relaxation when RBCs are subjected to periodic shear stress and deformation forces as they pass through an obstacle. We show that red blood cells are reversibly deformed and take on characteristic shapes not previously seen in physiological buffers when the maximum shear stress was between 2.2 and 25 Pa (strain rate 2200 to 25,000 s−1). We quantify the rates of RBC deformation and recovery using Kaplan–Meier survival analysis. The time to deformation decreased from 320 to 23 milliseconds with increasing flow rates, but the distance traveled before deformation changed little. Shape recovery, a measure of degree of deformation, takes tens of milliseconds at the lowest flow rates and reached saturation at 2.4 s at a shear stress of 11.2 Pa indicating a maximum degree of deformation was reached. The rates and types of deformation have relevance in red blood cell disorders and in blood cell behavior in microfluidic devices.

Summary We developed a universal method termed OnCELISA to detect cytokine secretion from individual cells by applying a capture technology on the cell membrane. OnCELISA uses fluorescent magnetic nanoparticles as assay reporters that enable detection on a single-cell level in microscopy and flow cytometry and fluorimetry in cell ensembles. This system is flexible and can be modified to detect different cytokines from a broad range of cytokine-secreting cells. Using OnCELISA we have been able to select and sort highly cytokine-secreting cells and identify cytokine-secreting expression profiles of different cell populations in vitro and ex vivo. We show that this system can be used for ultrasensitive monitoring of cytokines in the complex biological environment of atherosclerosis that contains multiple cell types. The ability to identify and select cell populations based on their cytokine expression characteristics is valuable in a host of applications that require the monitoring of disease progression., Graphical Abstract, Highlights • We developed an assay for detecting cytokine secretion from individual cells • This system is universal to detect different cytokines from a broad range of cell types • The assay can select highly cytokine-secreting cells and purify their populations • This study opens a host of applications for monitoring disease progression, Sensor; Biotechnology; Cell; Nanomaterials

Interferon-gamma (IFN-γ), a proinflammatory cytokine, has been used as an early indicator of multiple infectious diseases or tumors. In order to explore the detection capability of a commonly used anti-IFN-γ aptamer, a simple target induced strand-displacement aptasensing strategy was tested by introducing three different complementary strands and two different signal/quencher pairs. The Texas red/BHQ2-based sensor showed the best affinity constant (Kd) of 21.87 ng mL−1. It was found that the strand-displacement aptasensing strategy was impacted by the complementary position and length of the complementary strands. Additionally, the hybridization chain reaction (HCR) amplification strategy was introduced, yielding a 12-fold improved sensitivity of 0.45 ng mL−1. In order to further explore the sensing platform for spatially localized cytokine detection, the Texas red/BHQ2-based strand-displacement aptasensor was successfully fabricated on the 3D optical fiber surface to achieve a deployable sensing device for monitoring IFN-γ based on the fluorescence spots counting strategy. Finally, the three developed aptasensing strategies (strand-displacement strategy, HCR amplification strategy, 3D optical fiber aptasensor) were applied for detection of IFN-γ secreted by PBMCs with comparable results to those of ELISA. The deployable 3D optical fiber aptasensor with the superior sensitivity is potential to be used for detection of spatially localized IFN-γ in vivo.

A microfabricated needle-like probe has been designed and applied for hydrogen peroxide (H2O2) sampling and detection using a commercial, single-step fluorescent H2O2 assay. In this work, droplets of the assay reagent are generated and sent to the needle tip using a mineral-oil carrier fluid. At the needle tip, the sample is drawn into the device through 100 μm long hydrophilic capillaries by negative pressure. The sampled fluid is immediately merged with the assay droplet and carried away to mix and react, producing a sequence of droplets representing the H2O2 concentration as a function of time. We have characterized the assay fluorescence for small variations in the sample volume. With the calibration, we can calculate the concentration of H2O2 in the sampled liquid from the size and intensity of each merged droplet. This is a microfluidic data-logger system for on-site continuous sampling, controlled reaction, signal storage and on-line quantitative detection. It is a useful tool for monitoring dynamic chemical reactions in analytical chemistry and biological applications.

Microfluidic focusing of particles (both synthetic and biological), which enables precise control over the positions of particles in a tightly focused stream, is a prerequisite step for the downstream processing, such as detection, trapping and separation. In this study, we propose a novel hydrodynamic focusing method by taking advantage of open v-shaped microstructures on a glass substrate engraved by femtosecond pulse (fs) laser. The fs laser engraved microstructures were capable of focusing polystyrene particles and live cells in rectangular microchannels at relatively low Reynolds numbers (Re). Numerical simulations were performed to explain the mechanisms of particle focusing and experiments were carried out to investigate the effects of groove depth, groove number and flow rate on the performance of the groove-embedded microchannel for particle focusing. We found out that 10-µm polystyrene particles are directed toward the channel center under the effects of the groove-induced secondary flows in low-Re flows, e.g. Re

Deterministic Lateral Displacement (DLD) is a microfluidic method of separating particles by size. DLD relies on precise flow patterns to deliver highresolutionparticle separation. These patterns determine which particles are displacedlaterally, and which follow the flow direction. Prior research has demonstrated that the lateral array boundaries can be designed to improve the uniformity of the critical size and hence separation performance. A DLD device with an invariant critical size throughout is yet unknown. In this work we propose a 3D design approach. We first represent the flow through the DLD as a 2D lattice of resistors. This is used to determine the relative flow resistances at the boundaries that will deliver the correct flux patterns. We then use the Lattice Boltzmann Method to simulate fluid flow in a 3D unit cell of the DLD and measure the fluidic resistance for a range or typical dimensions. The results of this work are used to create a new equation for fluidic resistance as a function of post size, post height, and post spacing.We use this equation to determine array geometries that should have the appropriate resistances. We then design and simulate (in COMSOL) complete devices and measure fluid fluxes and first flow-lane widths along the boundaries. We find that the first flow-lane widths are much more uniform than in any devices described previously. This work providesthe best method for designing periodic boundaries, and enables narrower, shorter and higher throughput DLD devices.

Background Vascular targeting uses molecular markers on the surface of diseased vasculature for ligand-directed drug delivery to induce vessel occlusion or destruction. In the absence of discriminatory markers, such as in brain arteriovenous malformations (AVMs), stereotactic radiosurgery may be used to prime molecular changes on the endothelial surface. This study explored αB-crystallin (CRYAB) as a radiation induced target and pre-tested the specificity and efficacy of a CRYAB-targeting coaguligand for in vitro thrombus induction. Methods A parallel-plate flow system was established to circulate human whole blood over a layer of human brain endothelial cells. A conjugate of anti-CRYAB antibody and thrombin was injected into the circuit to compare binding and thrombus formation on cells with or without prior radiation treatment (0–25 Gy). Results Radiation increased CRYAB expression and surface exposure in human brain endothelial cells. In the parallel-plate flow system, the targeted anti-CRYAB–thrombin conjugate increased thrombus formation on the surface of irradiated cells relative to non-irradiated cells and to a non-targeting IgG-thrombin conjugate. Fibrin deposition and accumulation of fibrinogen degradation products increased significantly at radiation doses at or above 15 Gy with conjugate concentrations of 1.25 and 2.5 μg/mL. Conclusions CRYAB exposure can be detected at the surface of human brain endothelial cells in response to irradiation. Pro-thrombotic CRYAB-targeting conjugates can bind under high flow conditions and in the presence of whole blood induce stable thrombus formation with high specificity and efficacy on irradiated surfaces. CRYAB provides a novel radiation marker for potential vascular targeting in irradiated brain AVMs.

The advent of microfluidics in the 1990s promised a revolution in multiple industries from healthcare to chemical processing. Deterministic lateral displacement (DLD) is a continuous-flow microfluidic particle separation method discovered in 2004 that has been applied successfully and widely to the separation of blood cells, yeast, spores, bacteria, viruses, DNA, droplets, and more. Deterministic lateral displacement is conceptually simple and can deliver consistent performance over a wide range of flow rates and particle concentrations. Despite wide use and in-depth study, DLD has not yet been fully elucidated or optimized, with different approaches to the same problem yielding varying results. We endeavor here to provide up-to-date expert opinion on the state-of-art and current fundamental, practical, and commercial challenges with DLD as well as describe experimental and modeling opportunities. Because these challenges and opportunities arise from constraints on hydrodynamics, fabrication, and operation at the micro- and nanoscale, we expect this Perspective to serve as a guide for the broader micro- and nanofluidic community to identify and to address open questions in the field.

This paper concerns the design, fabrication and characterization of graphite/PDMS sensors for low-force sensing applications. Exploiting the design flexibility of 3D printing, moulds of specific dimensions were prepared onto which graphite powder and PDMS were cast, to develop sensor patches. The sensor patches were highly flexible with repeatable responses to iterative bending cycles. The patches were tested in terms of stretchability, strain and bending-cycle responses. The sensor patches had interdigitated electrodes operating on capacitive sensing, where the effective capacitance changes with an applied force because of changes in their dimensions. Forces ranging from 3.5 mN to 17.5 mN were applied to determine the capability of these sensor patches for low-force sensing applications. The sensor patches had a quick recovery having a sensitivity and SNR per unit force of 0.2542 pF mN−1 and 10.86 respectively. The patches were capable of differentiating the forces applied on them, when they were attached to different objects in daily use.

Reliable cell recovery and expansion are fundamental to the successful scale-up of chimeric antigen receptor (CAR) T cells or any therapeutic cell-manufacturing process. Here, we extend our previous work in whole blood by manufacturing a highly parallel deterministic lateral displacement (DLD) device incorporating diamond microposts and moving into processing, for the first time, apheresis blood products. This study demonstrates key metrics of cell recovery (80%) and platelet depletion (87%), and it shows that DLD T-cell preparations have high conversion to the T-central memory phenotype and expand well in culture, resulting in twofold greater central memory cells compared to Ficoll-Hypaque (Ficoll) and direct magnetic approaches. In addition, all samples processed by DLD converted to a majority T-central memory phenotype and did so with less variation, in stark contrast to Ficoll and direct magnetic prepared samples, which had partial conversion among all donors (

© 2018 Elsevier Ltd Background: Our goal is to develop a vascular targeting treatment for brain arteriovenous malformations (AVMs). Externalized phosphatidylserine has been established as a potential biomarker on the endothelium of irradiated AVM blood vessels. We hypothesize that phosphatidylserine could be selectively targeted after AVM radiosurgery with a ligand-directed vascular targeting agent to achieve localized thrombosis and rapid occlusion of pathological AVM vessels. Objective: The study aim was to establish an in vitro parallel-plate flow chamber to test the efficacy of a pro-thrombotic conjugate targeting phosphatidylserine. Methods: Conjugate was prepared by Lys-Lys cross-linking of thrombin with the phosphatidylserine-targeting ligand, annexin V. Cerebral microvascular endothelial cells were irradiated (5, 15, and 25 Gy) and after 1 or 3 days assembled in a parallel-plate flow chamber containing whole human blood and conjugate (1.25 or 2.5 μg/mL). Confocal microscopy was used to assess thrombus formation after flow via binding and aggregation of fluorescently-labelled platelets and fibrinogen. Results and conclusions: The annexin V-thrombin conjugate induced rapid thrombosis (fibrin deposition) on irradiated endothelial cells under shear stress in the parallel-plate flow device. Unconjugated, non-targeting thrombin did not induce fibrin deposition. A synergistic interaction between radiation and conjugate dose was observed. Thrombosis was greatest at the highest combined doses of radiation (25 Gy) and conjugate (2.5 μg/mL). The parallel-plate flow system provides a rapid method to pre-test pro-thrombotic vascular targeting agents. These findings validate the translation of the annexin V-thrombin conjugate to pre-clinical studies.

Microfluidics offer microenvironments for reagent delivery, handling, mixing, reaction, and detection, but often demand the affiliated equipment for liquid control for these functions. As a helpful tool, the capillary pressure control valve (CPCV) has become popular to avoid using affiliated equipment. Liquid can be handled in a controlled manner by using the bubble pressure effects. In this paper, we analyze and categorize the CPCVs via three determining parameters: surface tension, contact angle, and microchannel shape. Finally, a few application scenarios and impacts of CPCV are listed, which includes how CPVC simplify automation of microfluidic networks, work with other driving modes; make extensive use of microfluidics by open channel, and sampling and delivery with controlled manners. The authors hope this review will help the development and use of the CPCV in microfluidic fields in both research and industry.

Cytokines play critical roles in homeostatic control of health and they are integral for the creation and maintenance of a myriad of disease states. Their ultra-low concentration, often in the picomolar range, and extremely dynamic transient secretion process place stringent demands on cytokine quantification. We developed a nanoparticle-based strategy to detect trace cytokine secretion from individual, single live cells, for which we coined the term “OnCELISA”. Using a capture surface on the cell membrane and fluorescent magnetic nanoparticles as assay reporters, our universal OnCELISA assay achieved the sensitivity 0.1 pg mL-1, an over 10-fold enhancement, compared to state-of-the-art. The sensitive OnCELISA cell labelling made it possible to select and sort different cell types to determine highly cytokine - secreting cell subpopulations . The capture surfaces on cell membranes did not show noticeable effect on cell viability and their subsequent proliferation. The capability to specifically select such highly cytokine-secreting cells and purify their populations is pivotal for their use in multicellular pathologies such as atherosclerosis. Accordingly, we used this new approach to label cytokine secretion from vascular tissues of apolipoprotein E-/- mice; an in vivo model of atherosclerosis. In response to lipopolysaccharide, we observed increased capture of cytokine using this model. With the capacity of monitoring multiple cytokine secretions (IL-6 and IL-1β)), our OnCELISA method is able to probe how the individual cells and tissues secrete cytokines as they respond in real time to the surrounding signals.

In order to satisfy the need for sensitive detection of Aflatoxin M1 (AFM1), we constructed a simple and signal-on fluorescence aptasensor based on an autocatalytic Exonuclease III (Exo III)-assisted signal amplification strategy. In this sensor, the DNA hybridization on magnetic nanobeads could be triggered by the target AFM1, resulting in the release of a single-stranded DNA to induce an Exo III-assisted signal amplification, in which numerous G-quadruplex structures would be produced and then associated with the fluorescent dye to generate significantly amplified fluorescence signals resulting in the increased sensitivity. Under the optimized conditions, this aptasensor was able to detect AFM1 with a practical detection limit of 9.73 ng kg&minus, 1 in milk samples. Furthermore, the prepared sensor was successfully used for detection of AFM1 in the commercially available milk samples with the recovery percentages ranging from 80.13% to 108.67%. Also, the sensor performance was evaluated by the commercial immunoassay kit with satisfactory results.

In this work, we investigate methods of fabricating a device for the optical actuation of nanoparticles. To create the microfluidic channel, we pursued three fabrication methods: SU-8 to molded polydimethylsiloxane soft lithography, laser etching of glass, and deep reactive ion etching of fused silica. We measured the surface roughness of the etched sidewalls, and the laser power transmission through each device. We then measured the radiation pressure on 0.5-µm particles in the best-performing fabricated device (etched fused silica) and in a square glass capillary.

Gradient-based electrophoretic separations enable simultaneous separation and concentration of molecules. Compared with conventional injection-based separations, they enable enrichment of low-concentration analytes from larger sample volumes that are not limited by an injection volume. We have demonstrated that a nanochannel, connecting two chemically different reservoirs, can maintain a stationary chemical gradient while trapping biomolecules and effectively averaging out many of the complex physicochemical hydrodynamics that would broaden the bands in a meso- or microscale capillary. Here we describe chemical and physical methods that enable this work.

Gradient-based electrophoretic separations enable simultaneous separation and concentration of molecules. Compared with conventional injection-based separations, they enable enrichment of low-concentration analytes from larger sample volumes that are not limited by an injection volume. We have demonstrated that a nanochannel, connecting two chemically different reservoirs, can maintain a stationary chemical gradient while trapping biomolecules and effectively averaging out many of the complex physicochemical hydrodynamics that would broaden the bands in a meso- or microscale capillary. Here we describe chemical and physical methods that enable this work.

Droplet-based microfluidics are capable of transporting very small amounts of fluid over long distances. This characteristic may be applied to conventional fluid delivery using needles if droplets can be reliably expelled from a microfluidic channel. In this paper, we demonstrate a system for the extraction of water droplets from an oil-phase in a polymer microfluidic device. A hydrophilic membrane with a strong preference for water over oil is integrated into a droplet microfluidic system and observed to allow the passage of the transported aqueous phase droplets while blocking the continuous phase. The oil breakthrough pressure of the membrane was observed to be 250 ± 20 kPa, a much greater pressure than anywhere within the microfluidic channel, thereby eliminating the possibility that oil will leak from the microchannel, a critical parameter if droplet transport is to be used in needle-based drug delivery.

Micropost arrays serve as a plaform for the next generation of diagnostic devices. These arrays are found in microfluidic devices for peripheral blood-based diagnostics and metastatic cancer management. The function and performance of these devices is determined by the underlying micro-scale fluid mechanics. Typically, these devices operate in the creeping regime (Re << 1) where the viscous forces of the fluids dominate. Recent advances in manufacturing allow for higher Reynolds number flows (Re >> 1) where the inertial forces dominate. In this work, we use computational simulations to show there is a transitional region (1 < Re < 20) in between the laminar and creeping regimes for two different micropost array geometries. Numerical analysis is employed to investigate jet formation both within the array and at the array exit. The peak-to-peak amplitude of the streamwise normalized velocity profile is used to quantify jet formation within the array; the streamwise velocity profile at the end of the array exit is used to determine jet length at the exit of the array. Above the transitional region (Re > 20) significant jets form downstream of the posts, amplitude scales exponentially and jet length scales with Re according to power law.

Previous work has demonstrated the simultaneous concentration and separation of proteins via a stable ion concentration gradient established within a nanochannel (Inglis Angew. Chem., Int. Ed. 2001, 50, 7546-7550). To gain a better understanding of how this novel technique works, we here examine experimentally and numerically how the underlying electric potential controlled ion concentration gradients can be formed and controlled. Four nanochannel geometries are considered. Measured fluorescence profiles, a direct indicator of ion concentrations within the Tris-fluorescein buffer solution, closely match depth-averaged fluorescence profiles calculated from the simulations. The simulations include multiple reacting species within the fluid bulk and surface wall charge regulation whereby the deprotonation of silica-bound silanol groups is governed by the local pH. The three-dimensional system is simulated in two dimensions by averaging the governing equations across the (varying) nanochannel width, allowing accurate numerical results to be generated for the computationally challenging high aspect ratio nanochannel geometries. An electrokinetic circuit analysis is incorporated to directly relate the potential drop across the (simulated) nanochannel to that applied across the experimental chip device (which includes serially connected microchannels). The merit of the thick double layer, potential-controlled concentration gradient as a particle focusing and separation tool is discussed, linking this work to the previously presented protein trapping experiments. We explain why stable traps are formed when the flow is in the opposite direction to the concentration gradient, allowing particle separation near the low concentration end of the nanochannel. We predict that tapered, rather than straight nanochannels are better at separating particles of different electrophoretic mobilities.

We demonstrate matrix-free pH gradient electrofocusing of proteins within an 85 nm deep nanochannel. In contrast to conventional isoelectric focusing where the fluid does not move, this pH gradient method traps protein molecules flowing through a channel by balancing electric forces due to pH-dependent protein charge and viscous drag forces caused by electro-osmosis. The nanoscale depth of the device and the low voltage used limit convection relative to diffusion, thus producing a stable focused band of protein. R-Phycoerythrin (RPE) and Dylight labeled streptavidin (Dyl-Strep) were focused within a nanochannel using applied voltages between 0.4 and 1.6 V. Concentration enhancement factors of over 380 have been achieved within 5 min. Varying the buffer pH (between 2.7 and 7.2) at the boundaries of the nanochannel affected the shape of the focused bands. For RPE, a pH span of 4.5 (pH 2.7 to 7.2) yielded the narrowest peak while a span of 2.4 (pH 2.7 to 5.1) produced a significantly wider peak. Such matrix-free nanofluidic devices with pH gradient electrofocusing may enable on-chip integration of orthogonal separation techniques with mass spectrometry offering labor savings and enhanced performance.

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., 13 pages, 10 figures, 1 table, DLD, particle separation, microfluidics, anisotropic permeability

Automated and unbiased methods of non-invasive cell monitoring able to deal with complex biological heterogeneity are fundamentally important for biology and medicine. Label-free cell imaging provides information about endogenous fluorescent metabolites, enzymes and cofactors in cells. However extracting high content information from imaging of native fluorescence has been hitherto impossible. Here, we quantitatively characterise cell populations in different tissue types, live or fixed, by using novel image processing and a simple multispectral upgrade of a wide-field fluorescence microscope. Multispectral intrinsic fluorescence imaging was applied to patient olfactory neurosphere-derived cells, cell model of a human metabolic disease MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like syndrome). By using an endogenous source of contrast, subtle metabolic variations have been detected between living cells in their full morphological context which made it possible to distinguish healthy from diseased cells before and after therapy. Cellular maps of native fluorophores, flavins, bound and free NADH and retinoids unveiled subtle metabolic signatures and helped uncover significant cell subpopulations, in particular a subpopulation with compromised mitochondrial function. The versatility of our method is further illustrated by detecting genetic mutations in cancer, non-invasive monitoring of CD90 expression, label-free tracking of stem cell differentiation, identifying stem cell subpopulations with varying functional characteristics, tissue diagnostics in diabetes, and assessing the condition of preimplantation embryos. Our optimal discrimination approach enables statistical hypothesis testing and intuitive visualisations where previously undetectable differences become clearly apparent.

Automated and unbiased methods of non-invasive cell monitoring able to deal with complex biological heterogeneity are fundamentally important for biology and medicine. Label-free cell imaging provides information about endogenous autofluorescent metabolites, enzymes and cofactors in cells. However extracting high content information from autofluorescence imaging has been hitherto impossible. Here, we quantitatively characterise cell populations in different tissue types, live or fixed, by using novel image processing and a simple multispectral upgrade of a wide-field fluorescence microscope. Our optimal discrimination approach enables statistical hypothesis testing and intuitive visualisations where previously undetectable differences become clearly apparent. Label-free classifications are validated by the analysis of Classification Determinant (CD) antigen expression. The versatility of our method is illustrated by detecting genetic mutations in cancer, non-invasive monitoring of CD90 expression, label-free tracking of stem cell differentiation, identifying stem cell subpopulations with varying functional characteristics, tissue diagnostics in diabetes and assessing the condition of preimplantation embryos.

Conventional detection of pathogenic or other biological contamination relies on amplification of DNA using sequence-specific primers. Recent work in nanofluidics has shown very high concentration enhancement of biomolecules with some degree of simultaneous separation. This work demonstrates the combination of these two approaches by selectively concentrating a mobility-shifted hybridization product, potentially enabling rapid detection of rare DNA fragments such as highly specific 16S ribosomal DNA. We have performed conductivity gradient electrofocusing within nanofluidic channels and have shown concentration of hybridized peptide nucleic acids and DNA oligomers. We also show selectivity to single base-pair mismatch on 18-mer oligos. This approach may enable sensitive optical detection of small amounts of DNA.

Background and Objective The photobiological effect of laser light on cells and tissues originates from light absorption by endogenous chromophores and hence it depends on the wavelength of light source and cell type. Earlier studies regarding the biostimulation effects of green laser light investigated a wide variety of cells but not adipose tissue-derived stem cells (ADSCS). In this study we reported the in vitro effect of 532-nm Nd:YAG laser on proliferation, mitochondrial activity of these mesenchymal stem cells (MSCs) on the autofluorescence emission at wavelengths associated with nicotinamide adenine dinucleotide (NADH) and flavoproteins. Materials and Methods ADSCS were exposed to 532 nm second harmonic generation laser light at moderate power density (0.153 W/cm2) for periods of 30, 45, 60, 180, and 300 seconds. Mitochondrial membrane potential was measured using JC1 stain and confocal laser scanning microscopy, cell proliferation rates, and cellular autofluorescence emission at 450 and 540 nm wavelengths were measured using micro plate spectrofluorometer 48 hours after irradiation. Results Shorter (30–60 seconds) exposure times led to significantly increased proliferation, attributed to increased mitochondrial activity (P

An increasing demand in performance from electronic devices has resulted in continuous shrinking of electronic components. This shrinkage has demanded that the primary integration platform, the printed circuit board (PCB), follow this same trend. Today, PCB companies offer ~100 micron sized features (depth and width) which mean they are becoming suitable as physical platforms for Lab-on-a-Chip (LOC) and microfluidic applications. Compared to current lithographic based fluidic approaches; PCB technology offers several advantages that are useful for this technology. These include: Being easily designed and changed using free software, robust structures that can often be reused, chip layouts that can be ordered from commercial PCB suppliers at very low cost (1 AUD each in this work), and integration of electrodes at no additional cost. Here we present the application of PCB technology in connection with microfluidics for several biomedical applications. In case of commercialization the costs for each device can be even further decreased to approximately one tenth of its current cost.

Glass bonding is often necessary in microfluidic, nanofluidic and MEMS applications. Fusion bonding is a popular method in which glass substrates are permanently bonded using chemical treatment, low pressure and annealing. This project investigates a range of bonding methods and measures the bond strength. Float glass substrates were chemically cleaned and prepared using four different methods (Ethanol Rub, Ethanol Rub + NH4OH, piranha + NH4OH, and piranha + HCl + NH4OH), pressed together by hand, and placed in the oven at 400 °C for 10 h. Bond strength was assessed using compressive shear stress. It was determined that the piranha + HCl +NH4OH produced the strongest bond. All methods produced satisfactory bonds and the simplicity and safety of a simple Ethanol rub means it should be considered whenever possible.

We have developed a microfluidic needle-like device that can extract and deliver nanoliter samples. The device consists of a T-junction to form segmented flows, parallel channels to and from the needle tip, and seven hydrophilic capillaries at the tip that form a phase-extraction region. The main microchannel is hydrophobic and carries segmented flows of water-in-oil. The hydrophilic capillaries transport the aqueous phase with a nearly zero pressure gradient but require a pressure gradient of 19 kPa for mineral oil to invade and flow through. Using this device, we demonstrate the delivery of nanoliter droplets and demonstrate sampling through the formation of droplets at the tip of our device. During sampling, we recorded the fluorescence intensities of the droplets formed at the tip while varying the concentration of dye outside the tip. We measured a chemical signal response time of approximately 3 s. The linear relationship between the recorded fluorescence intensity of samples and the external dye co...

Urokinase plasminogen activator receptor (uPAR) and the epithelial integrin αvβ6 are thought to individually play critical roles in cancer metastasis. These observations have been highlighted by the recent discovery (by proteomics) of an interaction between these two molecules, which are also both implicated in the epithelial-mesenchymal transition (EMT) that facilitates escape of cells from tissue barriers and is a common signature of cancer metastases. In this study, orthogonal in cellulo and in vitro functional proteomic approaches were used to better characterize the uPAR·αvβ6 interaction. Proximity ligation assays (PLA) confirmed the uPAR·αvβ6 interaction on OVCA429 (ovarian cancer line) and four different colon cancer cell lines including positive controls in cells with de novo β6 subunit expression. PLA studies were then validated using peptide arrays, which also identified potential physical sites of uPAR interaction with αvβ6, as well as verifying interactions with other known uPAR ligands (e.g., uPA, vitronectin) and individual integrin subunits (i.e., αv, β1, β3, and β6 alone). Our data suggest that interaction with uPAR requires expression of the complete αβ heterodimer (e.g., αvβ6), not individual subunits (i.e., αv, β1, β3, or β6). Finally, using in silico structural analyses in concert with these functional proteomics studies, we propose and demonstrate that the most likely unique sites of interaction between αvβ6 and uPAR are located in uPAR domains II and III.

Isoelectric focusing of proteins in a silica nanofluidic channel filled with citric acid and disodium phosphate buffers is investigated via numerical simulation. Ions in the channel migrate in response to (i) the electric field acting on their charge and (ii) the bulk electroosmotic flow (which is directed toward the cathode). Proteins are focused near the low pH (anode) end when the electromigration effect is more significant and closer to the high pH (cathode) end when the electroosmotic effect dominates. We simulate the focusing behavior of Dylight labeled streptavidin (Dyl-Strep) proteins in the channel, using a relationship between the protein's charge and pH measured in a previous experiment. Protein focusing results compare well to previous experimental measurements. The effect of some key parameters, such as applied voltage, isoelectric point (pI), bulk pH, and bulk conductivity, on the protein trapping behavior in a nanofluidic channel is examined.