Microfluidic Array Research Articles

Long-term non-invasive interrogation of human dorsal root ganglion neuronal cultures on an integrated microfluidic multielectrode array platform.

Scientific studies in drug development and toxicology rely heavily on animal models, which often inaccurately predict the true response for human exposure. This may lead to unanticipated adverse effects or misidentified risks that result in, for example, drug candidate elimination. The utilization of human cells and tissues for in vitro physiological platforms has become a growing area of interest to bridge this gap and to more accurately predict human responses to drugs and toxins. The effects of new drugs and toxins on the peripheral nervous system are often investigated with neurons isolated from dorsal root ganglia (DRG), typically with one-time measurement techniques such as patch clamping. Here, we report the use of our multi-electrode array (MEA) platform for long-term noninvasive assessment of human DRG cell health and function. In this study, we acquired simultaneous optical and electrophysiological measurements from primary human DRG neurons upon chemical stimulation repeatedly through day in vitro (DIV) 23. Distinct chemical signatures were noted for the cellular responses evoked by each chemical stimulus. Additionally, the cell viability and function of the human DRG neurons were consistent through DIV 23. To the best of our knowledge, this is the first report on long-term measurements of the cell health and function of human DRG neurons on a MEA platform. Future generations will include higher electrode numbers in customized arrangements as well as integration with different tissue types on a single device. This platform will provide a valuable testing tool for both rodent and human cells, enabling a more comprehensive risk assessment for drug candidates and toxicants.

A microfluidic array for real-time live-cell imaging of human and rodent pancreatic islets.

In this study, we present a microfluidic array for high-resolution imaging of individual pancreatic islets. The device is based on hydrodynamic trapping principle and enables real-time analysis of islet cellular responses to insulin secretagogues. This device has significant advantages over our previously published perifusion chamber device including significantly increased analytical power and assay sensitivity, as well as improved spatiotemporal resolution. The islet array, with live-cell multiparametric imaging integration, provides a better tool to understand the physiological and pathophysiological changes of pancreatic islets through the analysis of single islet responses. This platform demonstrates the feasibility of array-based islet cellular analysis and opens up a new modality to conduct informative and quantitive evaluation of islets and cell-based screening for new diabetes treatments.

Highly efficient and gentle trapping of single cells in large microfluidic arrays for time-lapse experiments.

The isolation of single biological cells and their further cultivation in dedicated arrayed chambers are key to the collection of statistically reliable temporal data in cell-based biological experiments. In this work, we present a hydrodynamic single cell trapping and culturing platform that facilitates cell observation and experimentation using standard bio-lab equipment. The proposed design leverages the stochastic position of the cells as they flow into the structured microfluidic channels, where hundreds of single cells are then arrayed in nanoliter chambers for simultaneous cell specific data collection. Numerical simulation tools are used to devise and implement a hydrodynamic cell trapping mechanism that is minimally detrimental to the cell cycle and retains high overall trapping efficiency (∼70%) with the capability of reaching high fill factors (>90%) in short loading times (1-4 min) in a 400-trap device. A Monte Carlo model is developed using the design parameters to estimate the system trapping efficiencies, which show strong agreement with the experimentally acquired data. As proof of concept, arrayed mammalian tissue cells (MIA PaCa-2) are cultured in the microfluidic chambers for two days without viability problems.

Microfluidic hydrogel arrays for direct genotyping of clinical samples

A microfluidic hydrogel DNA microarray is developed to overcome the limitations of conventional planar microarrays such as low sensitivity, long overnight hybridization time, lack of a melting verification of proper hybrid, and complicated sample preparation process for genotyping of clinical samples. Unlike our previous prototype hydrogel array which can analyze only single-stranded DNA (ssDNA) targets, the device is the first of its type to allow direct multiplexed single nucleotide polymorphism (SNP) detection of human clinical samples comprising double-stranded DNA (dsDNA). This advance is made possible by incorporating a streptavidin (SA) hydrogel capture/purification element in a double T-junction at the start of the linear hydrogel array structure and fabricating ten different probe DNAs-entrapped hydrogels in microfluidic channels. The purified or unpurified polymerase chain reaction (PCR) products labeled with a fluorophore and a biotin are electrophoresed through the SA hydrogel for binding and purification. After electrophoretic washing, the fluorophore-labeled DNA strand is then thermally released for hybridization capture by its complementary probe gel element. We demonstrate the precise and rapid discrimination of the genotypes of five different clinical targets by melting curve analysis based on temperature-gradient electrophoresis within 3h, which is at least 3-fold decrease in incubation time compared to conventional microarrays. In addition, a 1.7pg (0.024 femtomoles) limit of detection for clinical samples is achieved which is ~100-fold better sensitivity than planar microarrays.

Automatic microfluidic fluorescence-array measurement system for detecting organic phosphate.

In this study, an automatic microfluidic fluorescence-array measurement system is developed to detect the concentration of organic phosphate based on the luminol-hydrogen peroxide catalytic fluorescent mechanism. Not only sample quantity and cost can be reduced, but also detection time, accuracy and precision can be improved in the system. The system is composed of a CCD image module, a stepper motor with driver, a microfluidic fluorescence array, a background light elimination module, and a dynamic image-analyzed interface. The pesticides of chlorpyrifos and fenitrothion of organic phosphate are chosen as experimental samples. Only a 2.5 μ l quantity of sample is required to have a fast response time of 1.4 second. Experimental results show that the sensitivities of chlorpyrifos and fenitrothion are 1.88 V/ppm in the range of 0.166 ∼ 10 ppm with averaged error of 1.66% and 0.32 V/ppm in the range of 0.03 ∼ 10 ppm with averaged error of 1.68% respectively. The organophosphorus effective detection range of the developed system covers the legal prescription for pesticide residues.

Apoptosis-related gene expression in tumor tissue samples obtained from patients diagnosed with glioblastoma multiforme.

Tumors of the brain are very diverse in their biological behavior and are therefore considered a major issue in modern medicine. The heterogeneity of gliomas, their clinical presentation and their responses to treatment makes this type of tumor a challenging area of research. Glioblastoma multiforme (GBM) is the most common, and biologically the most aggressive, primary brain tumor in adults. The standard treatment for patients with newly diagnosed GBM consists of surgical resection, radiotherapy and chemotherapy. However, resistance to chemotherapy is a major obstacle to successful treatment. The aim of this study was to examine the changes occurring in the expression levels of apoptosis-associated genes in tumor tissue biopsy samples from 7 patients diagnosed with GBM and compare our results with a human astrocyte cell line (used as a reference) cultured under basic conditions. For molecular analysis, we used a commercial pre-designed microfluidic array to quantify the expression of 93 apoptosis-associated human genes. Significant changes in the expression levels of genes were observed in the tumor tissue samples obtained from patients with GBM. We determined significant changes in gene expression (n=32) in all apoptotic signaling pathways (BCl-2, TNF, Caspases, NF-κB, IAP and CARD), while the most pronounced deregulation (>5-fold) were observed in 46.9% events. The results of this study underline the importance of apoptosis in heterogenous tumor tissue. The identification of the apoptotic gene panel in tissue biopsies from patients with GBM may help improve the effectiveness of treatments for GBM in clinical practice and may broaden our understanding of brain tumor cell metabolism. Recognizing the changes in the expression of pro-apoptotic and anti-apoptotic genes may aid in the development of novel treatment strategies founded on a molecular basis.

Towards doubling solar harvests using wide-angle, broad-band microfluidic beam steering arrays.

This paper introduces Microfluidic Beam Steering (MBS), which is a new technique for electronically steering light having multiple octaves of bandwidth, any polarization state and incidence from any direction of the sky without significant restrictions due to physical area, optical loss and power handling capacity. It is based on optical elements comprising both transparent solids and electronically controllable fluids to control Total Internal Reflection (TIR), refraction and/or diffraction from micro-structured surfaces within a transparent solid. A TIR-based MBS is discussed in the context of solar energy and its potential to significantly increase annual energy harvests from solar arrays situated on fixed areas like roofs. The advantages and challenges associated with analog and digital MBS systems are discussed and early-stage MBS hardware is demonstrated. Finally, an analytic model of sun-tracking is provided to formally establish the potential for MBS to increase annual solar energy harvests by approximately 45% more than conventional 0-Degree Of Freedom (0-DOF) solar arrays, 62% more than 1-DOF arrays and 233% more than 2-DOF arrays, all at 20% atmospheric aerosol scattering.

Fluorescence enhancement and multiple protein detection in ZnO nanostructure microfluidic devices.

In this study, different morphological ZnO nanostructures, those of sharp nanowires (NWs), rod NWs, and hexahedral-puncheon nanostructures, were grown in microfluidic channels on the same glass substrate. Characterizations of correspondent biomolecule binding properties were simulated and demonstrated. The surface was modified using 3-ammineopropyl-triethoxysilane (3-APTES) and biotin-N-hydroxysuccinimide ester (NHS-biotin). Different concentrations (4.17pM to 41.7nM) of dye-conjugated streptavidin were simultaneously infused through the second microfluidic channels, which lie 90° from the first microfluidic channels. The florescent intensity at the crossover areas showed good agreement with simulations, with sharp ZnO NWs exhibiting the largest dynamic range and the highest fluorescent intensity. We further characterize correspondent protein detection using sharp ZnO NWs. The surfaces of these ZnO NWs were modified with mouse immunoglobulin G (IgG), infused through the second microfluidic channels with dye-conjugated (Alexa 546) anti-mouse IgG in different concentrations. Concentrations ranging from 417fM to 41.7nM can be resolved using sharp ZnO NWs. Finally, multiple protein detection was demonstrated using a five-by-eight microfluidic channel array. Fluorescence images present clear multiple detections at the crossover areas when using the sharp ZnO NWs for simultaneous dye-conjugated anti-mouse IgG and dye-conjugated anti-rabbit IgG (Alexa 647) detection.

Passive Feed Network Designs for Microfluidic Beam-Scanning Focal Plane Arrays and Their Performance Evaluation

Microfluidic focal plane arrays (MFPAs) have been recently introduced to implement compact high-gain beam-scanning antennas without resorting to active RF devices. This beam-scanning technique relies on a patch antenna element that can be microfluidically repositioned at the focal plane of a microwave lens. The feed network is strategically designed to be passive and accommodate the position variation in the antenna element. This paper, for the first time, considers the design details and performance evaluation of three different passive network layouts that can potentially be utilized to excite MFPAs. Specifically, resonant corporate, resonant straight, and nonresonant straight microstrip line feed networks are introduced and their loss/bandwidth performances are investigated using the transmission line theory. In addition, a method of moments and ray tracing-based hybrid analysis is utilized to demonstrate the impact of the proposed feed networks on the radiation properties of the MFPA in terms of realized gain and side lobe levels (SLLs). It is shown that the resonant and nonresonant straight microstrip line feed networks reduce the SLL by more than 10 dB relative to the resonant corporate feed network utilized in the prior work. The performance improvements are experimentally verified through an eight-element extended hemispherical dielectric lens-based MFPA prototype. Different than the recent work that relied on liquid metal, the antenna element of this MFPA is implemented from a metalized plate by carrying out flow characterizations on various microfluidic channel geometries. This metalized plate approach paves the way for reliable liquid-metal-free microfluidic reconfigurable devices with higher efficiency and power-handling capabilities.

Rapid homogeneous endothelialization of high aspect ratio microvascular networks.

Microvascularization of an engineered tissue construct is necessary to ensure the nourishment and viability of the hosted cells. Microvascular constructs can be created by seeding the luminal surfaces of microfluidic channel arrays with endothelial cells. However, in a conventional flow-based system, the uniformity of endothelialization of such an engineered microvascular network is constrained by mass transfer of the cells through high length-to-diameter (L/D) aspect ratio microchannels. Moreover, given the inherent limitations of the initial seeding process to generate a uniform cell coating, the large surface-area-to-volume ratio of microfluidic systems demands long culture periods for the formation of confluent cellular microconduits. In this report, we describe the design of polydimethylsiloxane (PDMS) and poly(glycerol sebacate) (PGS) microvascular constructs with reentrant microchannels that facilitates rapid, spatially homogeneous endothelial cell seeding of a high L/D (2 cm/35 μm; > 550:1) aspect ratio microchannels. MEMS technology was employed for the fabrication of a monolithic, elastomeric, reentrant microvascular construct. Isotropic etching and PDMS micromolding yielded a near-cylindrical microvascular channel array. A 'stretch - seed - seal' operation was implemented for uniform incorporation of endothelial cells along the entire microvascular area of the construct yielding endothelialized microvascular networks in less than 24 h. The feasibility of this endothelialization strategy and the uniformity of cellularization were established using confocal microscope imaging.

In-Situ Characterization of Microfluidic Redox Battery with Dual-Pass Architecture

Microfluidic co-laminar flow cells is a growing field of energy research due to their inherent simplicity and low cost. In these cells, the two reactant containing electrolytes flow in a co-laminar manner that is dominated by slow diffusive mixing, which in turn forms an interface that provides the necessary separation of the reactant streams whilst allowing ion transfer between the electrodes [1]. Introducing the flow-through porous electrodes allowed higher performance in terms of power output and fuel utilization [2]. Furthermore, a unique dual-pass architecture was recently demonstrated which enabled in-situ recharging, fuel recirculation, and significant performance improvements [3-4]. The dual-pass cell architecture was further modified by splitting the electrodes, thus forming two symmetric cell portions, in order to experimentally measure and analyze the parasitic shunt current that appears in microfluidic electrochemical cell arrays with a shared electrolyte manifold [5-6]. In the present work, the same cell array design is used as an analytical device in order to analyze the performance characteristics of the dual-pass architecture and its application in co-laminar flow cells. The cell is fabricated using soft lithography techniques in polydimethylsiloxane (PDMS) using an SU-8 master and then bonded to a glass substrate. Porous carbon paper is cut into rectangular strips and placed as the electrodes for the device. Details about the vanadium redox electrolytes preparation and the device fabrication are described elsewhere [6]. Each cell portion is tested individually with vanadium redox species delivered at low and high flow rates and the results are used to quantify the species crossover losses, which result in a mixed potential and thus causes a drop in open circuit potential and overall performance. The crossover losses at the downstream portion are reduced from 41 mV at 10 μL/min to 13 mV at 100 μL/min. The upstream cell portion demonstrates maximum power density of 744 mW/cm2 at a high current density around 1000 mA/cm2. This compares favorably to all previously reported conventional counterparts. Moreover, the two cell portions are connected in parallel to resemble the original cell with dual-pass architecture [3] which allows assessing the contribution of the inlet and outlet passes of the dual-pass architecture in-situ. The fuel utilization is estimated from the current outputs of the two portions at low and high flow rates, and the contribution of the downstream cell portion is found to be on the same order as that of the upstream portion. Overall, the results of this study are expected to provide a deeper understanding of the reactant conversion and reactant crossover phenomena in co-laminar flow cells which will be useful for future device optimization. Acknowledgements Funding for this research provided by the Natural Sciences and Engineering Research Council of Canada (NSERC), Canada Foundation for Innovation (CFI), and British Columbia Knowledge Development Fund is highly appreciated.

Разработка микромашинных кибернетических платформ для культивирования эндотелиальных капиллярных сетей in vitro в пространстве организованных микропотоков питательной среды

This work is devoted to the development of technology and special equipment for the cultivation of spontaneously developing functioning endothelial capillary networks in vitro as the basis of artificial cloth-like structures with desired biological properties. It is the scientific and engineering projects RFBR №94-04-13544 «Structural analysis of microvascular bifurcations" and №96-04-50991 «Cell and Tissue Engineering endothelium (formation in endothelial culture in vitro the functioning self-developing capillary networks)." The proposed technology allows the author to form three-dimensional capillary endothelial network around micro-fluidic arrays, immersed in a specially designed dynamic gel. In 2013, the Korean research team under the lea-dership Noo Li Jeon has reproduced, using a similar approach, the phenomenon of self-developing functioning endothelial capillary networks with mass transfer in vitro. It has fully confirmed the validity of the concept pro-posed in the listed projects. Using system of the mathematical modeling Matlab & Simulink and system engi-neering design Cadence Orcad it was developed simulation mathematical model and circuit diagrams experimental reactor modules, it allows to saving considerable financial resources allocated to research and de-velopment of this kind. The resulting model contains 5.4 million basic Simulink blocks and performs more than 7,000 different mathematical functions, reflecting the behavior of devices in stationary and non-stationary conditions. Device control is based on neural network technology. Portable stand-alone microcomputers cyber platform includes microfluidic matrix, generators of microflows liquid phase nutrient medium, life-support systems of endothelial culture system of automatic digital imaging process of angiogenesis, the transmission system of encrypted data over a secure radio, digital control systems. All systems are backed up multiple times, allowing the product to operate in stand-alone mode for a long time (up to a year or more).

Ultrasensitive microfluidic array for serum pro-inflammatory cytokines and C-reactive protein to assess oral mucositis risk in cancer patients.

In addition to disease diagnostics, there is a need for biomarkers to predict severity of cancer therapy side effects such as oral mucositis. Oral mucositis is an inflammatory lesion of oral mucosa caused by high-dose chemotherapy and/or radiation that is especially prevalent during oral cancer treatment. We describe here a semi-automated, modular microfluidic immunoarray optimized for ultrasensitive detection of pro-inflammatory cytokines involved in pathobiology of oral mucositis. Our goal is to methodologically identify risk of mucositis early in oral cancer treatment, before the onset of lesions. Biomarkers include tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and C-reactive protein (CRP). Protein analytes were captured from serum in a capture chamber by 1-μm magnetic beads coated with antibodies and enzyme labels. Beads are then transported downstream to a detection chamber containing an eight-sensor array coated with glutathione-coated gold nanoparticles (GSH-AuNP) and a second set of antibodies to capture the beads with analyte proteins. In this first application of the immunoarray to four-protein multiplexed measurements, ultralow detection limits of 10-40 fg mL(-1) in 5 μL serum were achieved for simultaneous detection in 30 min. Mass detection limits were 2.5-10 zmol, as few as 1500 molecules. Accuracy and diagnostic utility of the arrays were demonstrated by correlation of levels of the four biomarker proteins in serum from head and neck cancer patients with results from standard ELISA. This approach may lead to rapid, low-cost estimates of projected risk for severity of oral mucositis in cancer patients to enable improved therapeutic management.

Housekeeping genes for studies of plasma microRNA: A need for more precise standardization

Plasma microRNAs (miRNAs) are promising biomarkers for many forms of cancer in humans; however, a fundamental concern is the lack of standardization in current data acquisition and reporting. Part of this problem lies in the use of numerous, different housekeeping genes (HKG) for the acquisition of real-time polymerase chain reaction data. This existing practice of using different HKGs generally is accepted, but reproducibility of data for comparison and validation between different laboratories calls for improvement. The need for data reproducibility standardization is crucial. An ideal plasma HKG (1) should be expressed in all samples, (2) have medium-to-high levels of expression, and (3) have consistently measurable levels of expression. Total RNA was extracted from 200-μL plasma samples via a modified miRNeasy (QIAGEN) extraction technique with yeast carrier. Total RNA purity was assessed with a Nanodrop 2000 spectrophotometer (Thermo Scientific). The cycle threshold (Ct) was fixed at 0.03 for all samples. We investigated 10 potential HKGs based both on reports in the literature and our previous data. The potential HKGs were Let-7a, Let-7d, Let-7g, miR-16, RNU6, RNU48, miR-191, miR-223, miR-484, and miR-520d-5p. Once all samples were run for each potential HKG, the mean Ct and SD was calculated for all sample groups, allowing for comparison among HKGs. We screened 380 miRNAs by using microfluidic array technology (Applied Biosystems) in a discovery cohort of 20 colorectal cancer (CRC) patients, 10 patients each with breast cancer (BC), lung cancer (LC), pancreatic cancer (PC), 11 patients with colorectal adenoma, and 12 controls. The mean Ct and SD was calculated for RNU6, miR-520d-5p, miR-16, miR-191, miR-223, and miR-484, which were expressed in all samples. Let-7a, Let-7d, Let-7g, and RNU48 were only expressed in 26%, 7%, 10%, and 8% of samples, respectively, and therefore were deemed to be insufficiently reliable HKGs. Only miRNAs with >50% expression were included in this statistical analysis. U6 and miR-520d-5p had the most consistent Ct as well as the least SD. The use of both RNU6 and 520d-5p as HKGs provided reliable results. Among HKGs that were expressed in all samples, we suggest that RNU6 and miR-520d-5p were the best candidates for HKGs for studies of plasma miRNA because of the consistent and high Ct in all samples and a very narrow, reproducible SD.

Recapitulation of in vivo-like paracrine signals of human mesenchymal stem cells for functional neuronal differentiation of human neural stem cells in a 3D microfluidic system

Paracrine signals produced from stem cells influence tissue regeneration by inducing the differentiation of endogenous stem or progenitor cells. However, many recent studies that have investigated paracrine signaling of stem cells have relied on either two-dimensional transwell systems or conditioned medium culture, neither of which provide optimal culture microenvironments for elucidating the effects of paracrine signals in vivo. In this study, we recapitulated in vivo-like paracrine signaling of human mesenchymal stem cells (hMSCs) to enhance functional neuronal differentiation of human neural stem cells (hNSCs) in three-dimensional (3D) extracellular matrices (ECMs) within a microfluidic array platform. In order to amplify paracrine signaling, hMSCs were genetically engineered using cationic polymer nanoparticles to overexpress glial cell-derived neurotrophic factor (GDNF). hNSCs were cultured in 3D ECM hydrogel used to fill central channels of the microfluidic device, while GDNF-overexpressing hMSCs (GDNF-hMSCs) were cultured in channels located on both sides of the central channel. This setup allowed for mimicking of paracrine signaling between genetically engineered hMSCs and endogenous hNSCs in the brain. Co-culture of hNSCs with GDNF-hMSCs in the 3D microfluidic system yielded reduced glial differentiation of hNSCs while significantly enhancing differentiation into neuronal cells including dopaminergic neurons. Neuronal cells produced from hNSCs differentiating in the presence of GDNF-hMSCs exhibited functional neuron-like electrophysiological features. The enhanced paracrine ability of GDNF-hMSCs was finally confirmed using an animal model of hypoxic-ischemic brain injury. This study demonstrates the presented 3D microfluidic array device can provide an efficient co-culture platform and provide an environment for paracrine signals from transplanted stem cells to control endogenous neuronal behaviors in vivo.

Numerical Study of Pillar Shapes in Deterministic Lateral Displacement Microfluidic Arrays for Spherical Particle Separation.

Deterministic lateral displacement (DLD) arrays containing shaped pillars have been found to be more effective in biomedical sample separation. This study aims to numerically investigate the interplay between particles and microfluidic arrays, and to find out the key factors in determining the critical size of a DLD device with shaped pillars. A new formula is thus proposed to estimate the critical size for spherical particle separation in this kind of new DLD microfluidic arrays. The simulation results show that both rectangular and I-shaped arrays have considerably smaller critical sizes. The ratio of sub-channel widths is also found to play an important role in reducing the critical sizes. This paves a valuable way toward designing high-performance DLD microfluidic arrays.

A transfer function approach for predicting rare cell capture microdevice performance.

Rare cells have the potential to improve our understanding of biological systems and the treatment of a variety of diseases; each of those applications requires a different balance of throughput, capture efficiency, and sample purity. Those challenges, coupled with the limited availability of patient samples and the costs of repeated design iterations, motivate the need for a robust set of engineering tools to optimize application-specific geometries. Here, we present a transfer function approach for predicting rare cell capture in microfluidic obstacle arrays. Existing computational fluid dynamics (CFD) tools are limited to simulating a subset of these arrays, owing to computational costs; a transfer function leverages the deterministic nature of cell transport in these arrays, extending limited CFD simulations into larger, more complicated geometries. We show that the transfer function approximation matches a full CFD simulation within 1.34 %, at a 74-fold reduction in computational cost. Taking advantage of these computational savings, we apply the transfer function simulations to simulate reversing array geometries that generate a "notch filter" effect, reducing the collision frequency of cells outside of a specified diameter range. We adapt the transfer function to study the effect of off-design boundary conditions (such as a clogged inlet in a microdevice) on overall performance. Finally, we have validated the transfer function's predictions for lateral displacement within the array using particle tracking and polystyrene beads in a microdevice.

Assay reproducibility in clinical studies of plasma miRNA.

There are increasing reports of plasma miRNAs as biomarkers of human disease but few standards in methodologic reporting, leading to inconsistent data. We systematically reviewed plasma miRNA studies published between July 2013-June 2014 to assess methodology. Six parameters were investigated: time to plasma extraction, methods of RNA extraction, type of miRNA, quantification, cycle threshold (Ct) setting, and methods of statistical analysis. We compared these data with a proposed standard methodologic technique. Beginning with initial screening for 380 miRNAs using microfluidic array technology and validation in an additional cohort of patients, we compared 11 miRNAs that exhibited differential expression between 16 patients with benign colorectal neoplasms (advanced adenomas) and 16 patients without any neoplasm (controls). Plasma was isolated immediately, 12, 24, 48, or 72 h following phlebotomy. miRNA was extracted using two different techniques (Trizol LS with pre-amplification or modified miRNeasy). We performed Taqman-based RT-PCR assays for the 11 miRNAs with subsequent analyses using a variable Ct setting or a fixed Ct set at 0.01, 0.03, 0.05, or 0.5. Assays were performed in duplicate by two different operators. RNU6 was the internal reference. Systematic review yielded 74 manuscripts meeting inclusion criteria. One manuscript (1.4%) documented all 6 methodological parameters, while < 5% of studies listed Ct setting. In our proposed standard technique, plasma extraction ≤12 h provided consistent ΔCt. miRNeasy extraction yielded higher miRNA concentrations and fewer non-expressed miRNAs compared to Trizol LS (1/704 miRNAs [0.14%] vs 109/704 miRNAs [15%], not expressed, respectively). A fixed Ct bar setting of 0.03 yielded the most reproducible data, provided that <10% miRNA were non-expressed. There was no significant intra-operator variability. There was significant inter-operator variation using Trizol LS extraction, while this was negligible using modified miRNeasy. For standardized reporting, we recommend plasma extraction ≤ 12 h, using modified miRNeasy extraction and utilizing a 0.03 Ct.

Simultaneous isolation and detection of circulating tumor cells with a microfluidic silicon-nanowire-array integrated with magnetic upconversion nanoprobes

The development of sensitive and convenient methods for detection, enrichment, and analysis of circulating tumor cells (CTCs), which serve as an importance diagnostic indicator for metastatic progression of cancer, has received tremendous attention in recent years. In this work, a new approach characteristic of simultaneous CTC capture and detection is developed by integrating a microfluidic silicon nanowire (SiNW) array with multifunctional magnetic upconversion nanoparticles (MUNPs). The MUNPs were conjugated with anti-EpCAM antibody, thus capable to specifically recognize tumor cells in the blood samples and pull them down under an external magnetic field. The capture efficiency of CTCs was further improved by the integration with a microfluidic SiNW array. Due to the autofluorescence free nature in upconversion luminescence (UCL) imaging, our approach allows for highly sensitive detection of small numbers of tumor cells, which afterward could be collected for further analysis and re-culturing. We have further demonstrated that this approach can be applied to detect CTCs in clinical blood samples from lung cancer patients, and obtained consistent results by analyzing the UCL signals and the clinical outcomes of lung cancer metastasis. Therefore our approach represents a promising platform in CTC capture and detection with potential clinical utilization in cancer diagnosis and prognosis.

Capture and X-ray diffraction studies of protein microcrystals in a microfluidic trap array.

X-ray free-electron lasers (XFELs) promise to enable the collection of interpretable diffraction data from samples that are refractory to data collection at synchrotron sources. At present, however, more efficient sample-delivery methods that minimize the consumption of microcrystalline material are needed to allow the application of XFEL sources to a wide range of challenging structural targets of biological importance. Here, a microfluidic chip is presented in which microcrystals can be captured at fixed, addressable points in a trap array from a small volume (<10 µl) of a pre-existing slurry grown off-chip. The device can be mounted on a standard goniostat for conducting diffraction experiments at room temperature without the need for flash-cooling. Proof-of-principle tests with a model system (hen egg-white lysozyme) demonstrated the high efficiency of the microfluidic approach for crystal harvesting, permitting the collection of sufficient data from only 265 single-crystal still images to permit determination and refinement of the structure of the protein. This work shows that microfluidic capture devices can be readily used to facilitate data collection from protein microcrystals grown in traditional laboratory formats, enabling analysis when cryopreservation is problematic or when only small numbers of crystals are available. Such microfluidic capture devices may also be useful for data collection at synchrotron sources.