Microfluidic Array Research Articles

Automated 3-D Printed Arrays to Evaluate Genotoxic Chemistry: E-Cigarettes and Water Samples.

A novel, automated, low cost, three-dimensional (3-D) printed microfluidic array was developed to detect DNA damage from metabolites of chemicals in environmental samples. The electrochemiluminescent (ECL) detection platform incorporates layer-by-layer (LbL) assembled films of microsomal enzymes, DNA and an ECL-emitting ruthenium metallopolymer in ∼10 nm deep microwells. Liquid samples are introduced into the array, metabolized by the human enzymes, products react with DNA if possible, and DNA damage is detected by ECL with a camera. Measurements of relative DNA damage by the array assess the genotoxic potential of the samples. The array analyzes three samples simultaneously in 5 min. Measurement of cigarette and e-cigarette smoke extracts and polluted water samples was used to establish proof of concept. Potentially genotoxic reactions from e-cigarette vapor similar to smoke from conventional cigarettes were demonstrated. Untreated wastewater showed a high genotoxic potential compared to negligible values for treated wastewater from a pollution control treatment plant. Reactivity of chemicals known to produce high rates of metabolite-related DNA damage were measured, and array results for environmental samples were expressed in terms of equivalent responses from these standards to assess severity of possible DNA damage. Genotoxic assessment of wastewater samples during processing also highlighted future on-site monitoring applications.

Use of a highly parallel microfluidic flow cell array to determine therapeutic drug dose response curves.

A high-throughput, microfluidic flow cell array (MFCA) system has been modified to enable drug screening against small-volume cell-, and tissue cultures. The MFCA is composed of a 3D channel network that simultaneously flows fluids through forty-eight 830μm by 500μm flow cells, which physically divide and fluidically seal an existing culture into multiple compartments when docked onto the surface of a cell or tissue culture dish. The modified system provides temperature (37°C) and CO2/pH level controls, while continuously flowing solutions (media or other liquid such as drug suspensions) over the cells/tissues. These assays were enhanced and validated using inverted microscopy and fluorescent staining techniques which also allow real time viability and toxicity assessments. This work presents the results of this new generation in vitro drug testing assay performed using this modified MFCA system. This setup allows the testing of 48 drug combinations on 48 different cell-, tissue specimen at once under flow conditions. All 48 flow cells were utilized to test 5 different concentrations of cisplatin (CDDP). CDDP solutions in various concentrations were continually flowed over cultured human ovarian cancer cells for 48h. Viability assessments were performed using red-orange calcein and SYTOX ® Green nucleic acid stains. Cells were imaged at the beginning and end of the experiment (48h). In order to compare and validate MFCAs suitability as drug screening assay, MTT assays were performed on cells. We found that both, MTT and MFCA assays generated dose-response curves with similar profiles. Innovative advantages of the MFCA system include the ability of handling smaller amounts of solutions compared to conventional and current state of the art drug screening and cell viability/toxicity methods. It also provides the ability to continually deliver fresh solution to the cell samples, while eliminating wastes that are produced. Based on our here reported findings MFCA may have a strong potential of providing a more physiological model than current state of the art static MTT assays.

Modern Approaches to Chemical Toxicity Screening.

Chemical toxicity has a serious impact on public health, and toxicity failures of drug candidates drive up drug development costs. Many in vitro bioassays exist for toxicity screening, and newer versions of these tend to be high throughput or high content assays, some of which rely on electrochemical detection. Toxicity very often results from metabolites of the chemicals we are exposed to, so it is important that assays feature metabolic conversion. Combining bioassays, computational predictions, and accurate chemical pathway elucidation presents our best chance for reliable toxicity prediction. Employing electrochemical and electrochemiluminescent approaches, cell-free microfluidic arrays can measure relative rates of formation of DNA-metabolite adduct formation (a measure of genotoxicity) as well as DNA oxidation levels resulting from enzyme-generated metabolites. Enzymes for several organ types can be studied simultaneously. These arrays can be used to identify the most reactive metabolites, and subsequent mechanistic details can then be investigated with high throughput LC-HPLC using enzyme/DNA-coated magnetic beads.

Microfluidic metamaterial sensor: Selective trapping and remote sensing of microparticles

We experimentally demonstrate the integration of a microfluidic trap array on top of metamaterial resonators for size selective trapping and remote sensing of microparticles. A split-ring resonator (SRR) design supports strongly confined electric field in the capacitive split gap at the fundamental inductive-capacitive resonance mode. The tightly confined electric field in the SRR gap forms a hot-spot that has become an enabling platform for sensing applications. Here, we extend the concept of metamaterial sensing to “trapping and sensing” by fabricating trapezoidal shaped structures near the split gap that enables trapping of microparticles in the split-gap region of each SRR. The proposed microfluidic metamaterial sensor enables sensing of different refractive index microparticles in terms of change in the transmitted amplitude and resonance frequency of the fundamental resonance mode operating in the terahertz spectral region. The proposed approach exploits the advantages offered by microfluidics, metamaterials, and terahertz technologies to form an ideal platform for ultra-sensitive, label-free, remote, and non-destructive detection of micro-substances.

In situ mRNA isolation from a microfluidic single-cell array using an external AFM nanoprobe.

We present an in situ mRNA extraction platform to quantify marker-genes' expression levels of single target cells within high-density microfluidic trapping arrays. This platform enables single-cell transcriptomic analysis to reveal in-depth information of cellular mechanisms and population heterogeneity. Although microfluidic technology enables the automation of single-cell sorting, trapping and identification, most developed microfluidic devices are closed off and prevent single-cell access by external analytical equipment. Besides, cell lysing is usually required for mRNA extraction. In our platform, cells are trapped individually in a microwell array sealed by a 1 μm-thick polydimethylsiloxane (PDMS) membrane, and a modified atomic force microscopy (AFM) probe-a dielectrophoretic nanotweezer (DENT)-penetrates through the membrane and extracts mRNA molecules from a single cell by dielectrophoresis. The single-cellular expression levels of 3 housekeeping genes from HeLa cells were analyzed quantitatively based on the quantification of the extracted mRNAs, and the probed cells remained viable when the applied alternating-current (AC) voltage was lower than 1.5 Vpp during mRNA probing. We also performed in situ mRNA isolation from a mixture of SK-BR-3 and U937 cells, mimicking a blood sample that underwent primary enrichment of circulating tumor cells (CTCs), and evaluated various marker-genes' expressions. This integrated platform combines the non-destructive and precise-control of a single-cell mRNA probe with sealed microfluidic systems' capability of upstream sample processing and downstream multifunctional analysis to enable a versatile and powerful tool for biomedical research.

Anisotropic permeability in deterministic lateral displacement arrays.

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.

Microfluidic channel-coupled 3D quartz nanohole arrays for high capture and release efficiency of BT20 cancer cells.

Nanostructured materials, such as silicon nanowires, quartz nanostructures, and polymer-modified nanostructures, are a promising new class of materials for the capture and enumeration of very rare tumor cells, including circulating tumor cells (CTCs), to examine their biological characteristics in whole blood of cancer patients. These cells can then be used for transplantation, anti-tumor cell therapy, and cell-secreted protein studies. It is believed that 3-dimensional (3D) nanostructured substrates efficiently enhance cell capture yields due to the increased local contacts between the 3D nanostructures and extracellular extensions of the tumor cells. Recent studies have been performed with enhanced cell capture yields thanks to various nanostructured platforms; however, there remains an urgent need both to capture and release viable rare tumor cells for further molecular (i.e., protein) analysis and to develop patient-specific drugs. Here, we first demonstrate that our 3D quartz nanohole array (QNHA) tumor cell capture and release system allows us not only to selectively capture rare tumor cells, but also to release the cells with high capture and release rates. This system was developed using streptavidin (STR)-functionalized QNHA (STR-QNHA) with a microfluidic channel. Our system has ideal cell-separation yields of as high as 85-91% and high release rates of >90% for the BT20 cell line. We suggest that the use of a microfluidic channel technique coupled with a STR-QNHA cell capture and release chip (STR-QNHA cell chip) would be a powerful and simple process to evaluate the capture, enumeration, and release of CTCs from patient whole blood for studying further cell therapy and tumor-cell-secreted molecules.

Multi-chamber microfluidic platform for high-precision skin permeation testing.

The established in vitro tool used for testing the absorption and penetration of chemicals through skin in pharmacology, toxicology and cosmetic science is the static Franz diffusion cell. While widespread, Franz cells are relatively costly, low-throughput and results may suffer from poor reproducibility. Microfluidics has the potential to overcome these drawbacks. In this paper, we present a novel microfluidic skin permeation platform and validate it rigorously against the Franz cell by comparing the transport of 3 model chemicals of varying lipophilicity: caffeine, salicylic acid and testosterone. Permeation experiments through silicone membranes show that the chip yields higher sensitivity in permeant cumulative amounts and comparable or lower coefficients of variation. Using a skin organotypic culture, we show that the chip decreases the effect of unstirred water layers that can occur in static Franz cells. The validation reported herein sets the stage for efficient skin permeation and toxicity screening and further development of microfluidic skin-on-chip devices.

A microfluidic trap array for longitudinal monitoring and multi-modal phenotypic analysis of individual stem cell aggregates.

Three-dimensional pluripotent stem cell (PSC) cultures have the ability to undergo differentiation, self-organization, and morphogenesis to yield complex, in vitro tissue models that recapitulate key elements of native tissues. These tissue models offer a system for studying mechanisms of tissue development, investigating disease mechanisms, and performing drug screening. It remains challenging, however, to standardize PSC aggregate differentiation and morphogenesis methods due to heterogeneity stemming from biological and environmental sources. It is also difficult to monitor and assess large numbers of individual samples longitudinally throughout culture using typical batch-based culture methods. To address these challenges, we have developed a microfluidic platform for culture, longitudinal monitoring, and phenotypic analysis of individual stem cell aggregates. This platform uses a hydrodynamic loading principle to capture pre-formed stem cell aggregates in independent traps. We demonstrated that multi-day culture of aggregates in this platform reduces heterogeneity in phenotypic parameters such as size and morphology. Additionally, we showed that culture and analysis steps can be performed sequentially in the same platform, enabling correlation of multiple modes of analysis for individual samples. We anticipate this platform being applied to improve abilities for phenotypic analysis of PSC aggregate tissues and to facilitate research in standardizing culture systems in order to dually increase the yield and reduce the heterogeneity of PSC-derived tissues.

Alterations in micro RNA-messenger RNA (miRNA-mRNA) Coupled Signaling Networks in Sporadic Alzheimer's Disease (AD) Hippocampal CA1.

RNA sequencing, DNA microfluidic array, LED-Northern, Western immunoassay and bioinformatics analysis have uncovered a small family of up-regulated human brain enriched microRNAs (miRNAs) and down-regulated messenger RNAs (mRNAs) in short post-mortem interval (PMI) sporadic Alzheimer’s disease (AD) brain. At the mRNA level, a large majority of the expression of human brain genes found to be down-regulated in sporadic AD appears to be a consequence of an up-regulation of a specific group of NF-kB-inducible microRNAs (miRNAs). This group of up-regulated miRNAs – including miRNA-34a and miRNA-146a - has strong, energetically favorable, complimentary RNA sequences in the 3′ untranslated regions (3′-UTR) of their target mRNAs which ultimately drive the down-regulation in the expression of certain essential brain genes. Interestingly, just 2 significantly up-regulated miRNAs - miRNA-34a and miRNA-146a – appear to down-regulate mRNA targets involved in synaptogenesis (SHANK3), phagocytosis deficits and tau pathology (TREM2), inflammation (CFH; complement factor H) and amyloidogenesis (TSPAN12), all of which are distinguishing pathological features characteristic of middle-to-late stage AD neuropathology. This paper reports the novel finding of parallel miRNA-34a and miRNA-146a up-regulation in sporadic AD hippocampal CA1 RNA pools and proposes an altered miRNA-mRNA coupled signaling network in AD, much of which is supported by current experimental findings in the recent literature.

Reduced Innate Immune Response to a Staphylococcus aureus Small Colony Variant Compared to Its Wild-Type Parent Strain.

Background: Staphylococcus aureus (S. aureus) small colony variants (SCVs) can survive within the host intracellular milieu and are associated with chronic relapsing infections. However, it is unknown whether host invasion rates and immune responses differ between SCVs and their wild-type counterparts. This study used a stable S. aureus SCV (WCH-SK2SCV) developed from a clinical isolate (WCH-SK2WT) in inflammation-relevant conditions. Intracellular infection rates as well as host immune responses to WCH-SK2WT and WCH-SK2SCV infections were investigated.Method: NuLi-1 cells were infected with either WCH-SK2WT or WCH-SK2SCV, and the intracellular infection rate was determined over time. mRNA expression of cells infected with each strain intra- and extra-cellularly was analyzed using a microfluidic qPCR array to generate an expression profile of thirty-nine genes involved in the host immune response.Results: No difference was found in the intracellular infection rate between WCH-SK2WT and WCH-SK2SCV. Whereas, extracellular infection induced a robust pro-inflammatory response, intracellular infection elicited a modest response. Intracellular WCH-SK2WT infection induced mRNA expression of TLR2, pro-inflammatory cytokines (IL1B, IL6, and IL12) and tissue remodeling factors (MMP9). In contrast, intracellular WCH-SK2SCV infection induced up regulation of only TLR2.Conclusions: Whereas, host intracellular infection rates of WCH-SK2SCV and WCH-SK2WT were similar, WCH-SK2SCV intracellular infection induced a less widespread up regulation of pro-inflammatory and tissue remodeling factors in comparison to intracellular WCH-SK2WT infection. These findings support the current view that SCVs are able to evade host immune detection to allow their own survival.

Next-generation nanotech meds: Diagnostic and therapeutic applications of non-organic nanoparticles are making their way into clinical use.

Nanoparticles have been used for more than a decade in medicine, one of the more prominent applications being silver particles in wound dressing to prevent infection and local inflammation. Most nanoparticles though are used for drug delivery, a substantial and fast growing market, worth about US$41 billion in 2014 and projected to reach US$118 billion by 2023 (http://www.transparencymarketresearch.com/nanotechnology-drug-delivery.html). In most cases, these particles are made of organic molecules to encapsulate therapeutic compounds or to home in on specific cell types such cancer cells. Recently, though, research has taken a growing interest in inorganic nanoparticles and early clinical trials are underway or about to begin soon. The potential applications of inorganic nanoparticles include diagnosis, therapy, or a combination of both for a wide range of degenerative, oncological, infectious and auto‐immune or inflammatory diseases. > … Nanoparticles often have properties that are different from the same elements or compounds at larger scale… Nanoparticles are usually defined as anything in size between 1 and 100 nm, which is just the right size for targeted delivery and diagnostics at the level of biomolecules. Moreover, nanoparticles often have properties that are different from the same elements or compounds at larger scale, while their large surface to volume ratio increases chemical reactivity and thus potential as catalysts. By the same token, this increases potential for deleterious side effects, which has so far held back the use of nanomaterials in the clinic. The most widely researched nanoparticles, particularly for applications in biology and medicine, are carbon nanotubes, gold nanoparticles, and cadmium selenide quantum dots. To a large extent, the properties that make these particles attractive for industrial applications are equally relevant in biology. Gold nanoparticles, for example, are highly flexible in terms of size, shape, surface chemistry, and aggregation state. Quantum dots can deliver high doses of energy …

High-throughput gene-expression quantification of grapevine defense responses in the field using microfluidic dynamic arrays.

BackgroundThe fight against grapevine diseases due to biotrophic pathogens usually requires the massive use of chemical fungicides with harmful environmental effects. An alternative strategy could be the use of compounds able to stimulate plant immune responses which significantly limit the development of pathogens in laboratory conditions. However, the efficiency of this strategy in natura is still insufficient to be included in pest management programs. To understand and to improve the mode of action of plant defense stimulators in the field, it is essential to develop reliable tools that describe the resistance status of the plant upon treatment.ResultsWe have developed a pioneering tool (“NeoViGen96” chip) based on a microfluidic dynamic array platform allowing the expression profiling of 85 defense-related grapevine genes in 90 cDNA preparations in a 4 h single run. Two defense inducers, benzothiadiazole (BTH) and fosetyl-aluminum (FOS), have been tested in natura using the “NeoViGen96” chip as well as their efficacy against downy mildew.BTH-induced grapevine resistance is accompanied by the induction of PR protein genes (PR1, PR2 and PR3), genes coding key enzymes in the phenylpropanoid pathway (PAL and STS), a GST gene coding an enzyme involved in the redox status and an ACC gene involved in the ethylene pathway.FOS, a phosphonate known to possess a toxic activity against pathogens and an inducing effect on defense genes provided a better grapevine protection than BTH. Its mode of action was probably strictly due to its fungicide effect at high concentrations because treatment did not induce significant change in the expression level of selected defense-related genes.ConclusionsThe NeoViGen96” chip assesses the effectiveness of plant defense inducers on grapevine in vineyard with an excellent reproducibility. A single run with this system (4 h and 1,500 €), corresponds to 180 qPCR plates with conventional Q-PCR assays (Stragene system, 270 h and 9,000 €) thus a throughput 60–70 times higher and 6 times cheaper. Grapevine responses after BTH elicitation in the vineyard were similar to those obtained in laboratory conditions, whereas our results suggest that the protective effect of FOS against downy mildew in the vineyard was only due to its fungicide activity since no activity on plant defense genes was observed. This tool provides better understanding of how the grapevine replies to elicitation in its natural environment and how the elicitor potential can be used to reduce chemical fungicide inputs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3304-z) contains supplementary material, which is available to authorized users.

Electro-Deformation of Fused Cells in a Microfluidic Array Device.

We present a new method of analyzing the deformability of fused cells in a microfluidic array device. Electrical stresses—generated by applying voltages (4–20 V) across discrete co-planar microelectrodes along the side walls of a microfluidic channel—have been used to electro-deform fused and unfused stem cells. Under an electro-deformation force induced by applying an alternating current (AC) signal, we observed significant electro-deformation phenomena. The experimental results show that the fused stem cells were stiffer than the unfused stem cells at a relatively low voltage (<16 V). However, at a relatively high voltage, the fused stem cells were more easily deformed than were the unfused stem cells. In addition, the electro-deformation process is modeled based on the Maxwell stress tensor and structural mechanics of cells. The theoretical results show that a positive correlation is found between the deformation of the cell and the applied voltage, which is consistent with the experimental results. Combined with a numerical analysis and experimental study, the results showed that the significant difference of the deformation ratio of the fused and unfused cells is not due to their size difference. This demonstrates that some other properties of cell membranes (such as the membrane structure) were also changed in the electrofusion process, in addition to the size modification of that process.

Microfluidic profiling of apoptosis-related genes after treatment with BH3-mimetic agents in astrocyte and glioblastoma cell lines.

Glioblastoma(GB) is the most frequent and biologically the most aggressive primary brain tumor in adults. Standard treatment for newly diagnosed GB consists of surgical resection, radiotherapy and chemotherapy. Resistance to therapy is a major obstacle, even with optimal treatment with a survival median of only 12-15 months. The heterogeneity and treatment response of GB makes this tumor type a challenging area of research. The aim of our study was to study the response of normal human astrocyte(HA) and human GB (T98G) cell lines to apoptosis inhibitors invitro. ABT-737 is an inhibitor of anti-apoptotic proteins Bcl-2, Bcl-xL, Bcl-w, while MIM-1 is an Mcl-1 protein inhibitor. The viability of the cells was assayed biochemically using the cytotoxic methyl thiazolyl tetrazolium(MTT) assay. Changes in the expression of apoptosis-associated genes (n=93) in two human brain cell lines after treatment with the apoptosis inhibitors ABT-737 and MIM-1 (individually), between the apoptosis inhibitor treated group and the control group, were determined using a commercially pre-designed microfluidic array. Significant changes in apoptotic gene expression with more than a 2.0-fold difference in their expression levels were obtained in both cell lines; the most altered genes were in the HA cell line after MIM-1 treatment (n=42). These results contribute to the importance of apoptosis in normal and cancerous brain tissues and provide information on the effect of apoptosis inhibitors on cell viability and gene expression. Despite extensive investigations, a cure for GB is currently not available. The identification of an apoptotic gene panel and determining the sensitivity of normal and GB brain cells to individual apoptosis inhibitors could help to improve clinical practice and increase our understanding of brain tumor cell metabolism and apoptosis inhibitors in GB cells and astrocytes. Recognizing expression changes in pro-apoptotic and anti-apoptotic genes could contribute to the development of new treatments.

A Highly Predictive Model for Diagnosis of Colorectal Neoplasms Using Plasma MicroRNA: Improving Specificity and Sensitivity.

To develop a plasma-based microRNA (miRNA) diagnostic assay specific for colorectal neoplasms, building upon our prior work. Colorectal neoplasms [colorectal cancer (CRC) and colorectal advanced adenoma (CAA)] frequently develop in individuals at ages when other common cancers also occur. Current screening methods lack sensitivity, specificity, and have poor patient compliance. Plasma was screened for 380 miRNAs using microfluidic array technology from a "Training" cohort of 60 patients, (10 each) control, CRC, CAA, breast cancer, pancreatic cancer, and lung cancer. We identified uniquely dysregulated miRNAs specific for colorectal neoplasia (P < 0.05, false discovery rate: 5%, adjusted α = 0.0038). These miRNAs were evaluated using single assays in a "Test" cohort of 120 patients. A mathematical model was developed to predict blinded sample identity in a 150 patient "Validation" cohort using repeat-sub-sampling validation of the testing dataset with 1000 iterations each to assess model detection accuracy. Seven miRNAs (miR-21, miR-29c, miR-122, miR-192, miR-346, miR-372, and miR-374a) were selected based upon P value, area under the curve (AUC), fold change, and biological plausibility. Area under the curve (±95% confidence interval) for "Test" cohort comparisons were 0.91 (0.85-0.96) between all neoplasia and controls, 0.79 (0.70-0.88) between colorectal neoplasia and other cancers, and 0.98 (0.96-1.0) between CRC and colorectal adenomas. In our "Validation" cohort, our mathematical model predicted blinded sample identity with 69% to 77% accuracy, 67% to 76% accuracy, and 86% to 90% accuracy for each comparison, respectively. Our plasma miRNA assay and prediction model differentiate colorectal neoplasia from patients with other neoplasms and from controls with higher sensitivity and specificity compared with current clinical standards.

Mechanically activated artificial cell by using microfluidics.

All living organisms sense mechanical forces. Engineering mechanosensitive artificial cell through bottom-up in vitro reconstitution offers a way to understand how mixtures of macromolecules assemble and organize into a complex system that responds to forces. We use stable double emulsion droplets (aqueous/oil/aqueous) to prototype mechanosensitive artificial cells. In order to demonstrate mechanosensation in artificial cells, we develop a novel microfluidic device that is capable of trapping double emulsions into designated chambers, followed by compression and aspiration in a parallel manner. The microfluidic device is fabricated using multilayer soft lithography technology, and consists of a control layer and a deformable flow channel. Deflections of the PDMS membrane above the main microfluidic flow channels and trapping chamber array are independently regulated pneumatically by two sets of integrated microfluidic valves. We successfully compress and aspirate the double emulsions, which result in transient increase and permanent decrease in oil thickness, respectively. Finally, we demonstrate the influx of calcium ions as a response of our mechanically activated artificial cell through thinning of oil. The development of a microfluidic device to mechanically activate artificial cells creates new opportunities in force-activated synthetic biology.

Dynamic analysis of immune and cancer cell interactions at single cell level in microfluidic droplets.

Cell-cell communication mediates immune responses to physiological stimuli at local and systemic levels. Intercellular communication occurs via a direct contact between cells as well as by secretory contact-independent mechanisms. However, there are few existing methods that allow quantitative resolution of contact-dependent and independent cellular processes in a rapid, precisely controlled, and dynamic format. This study utilizes a high-throughput microfluidic droplet array platform to analyze cell-cell interaction and effector functions at single cell level. Controlled encapsulation of distinct heterotypic cell pairs was achieved in a single-step cell loading process. Dynamic analysis of dendritic cell (DC)-T cell interactions demonstrated marked heterogeneity in the type of contact and duration. Non-stimulated DCs and T cells interacted less frequently and more transiently while antigen and chemokine-loaded DCs and T cells depicted highly stable interactions in addition to transient and sequential contact. The effector function of CD8+ T cells was assessed via cytolysis of multiple myeloma cell line. Variable cell conjugation periods and killing time were detected irrespective of the activation of T cells, although activated T cells delivered significantly higher cytotoxicity. T cell alloreactivity against the target cells was partially mediated by secretion of interferon gamma, which was abrogated by the addition of a neutralizing antibody. These results suggest that the droplet array-based microfluidic platform is a powerful technique for dynamic phenotypic screening and potentially applicable for evaluation of novel cell-based immunotherapeutic agents.

A pumpless microfluidic device driven by surface tension for pancreatic islet analysis.

We present a novel pumpless microfluidic array driven by surface tension for studying the physiology of pancreatic islets of Langerhans. Efficient fluid flow in the array is achieved by surface tension-generated pressure as a result of inlet and outlet size differences. Flow properties are characterized in numerical simulation and further confirmed by experimental measurements. Using this device, we perform a set of biological assays, which include real-time fluorescent imaging and insulin secretion kinetics for both mouse and human islets. Our results demonstrate that this system not only drastically simplifies previously published experimental protocols for islet study by eliminating the need for external pumps/tubing and reducing the volume of solution consumption, but it also achieves a higher analytical spatiotemporal resolution due to efficient flow exchanges and the extremely small volume of solutions required. Overall, the microfluidic platform presented can be used as a potential powerful tool for understanding islet physiology, antidiabetic drug development, and islet transplantation.

High-Throughput Electrochemical Microfluidic Immunoarray for Multiplexed Detection of Cancer Biomarker Proteins.

Microchip-based microfluidic electrochemical arrays hold great promise for fast, high-throughput multiplexed detection of cancer biomarker proteins at low cost per assay using relatively simple instrumentation. Here we describe an inexpensive high-throughput electrochemical array featuring 32 individually addressable microelectrodes that is further multiplexed with an 8-port manifold to provide 256 sensors. The gold electrode arrays were fabricated by wet-etching commercial gold compact discs (CD-R) followed by patterned insulation. A print-and-peel method was used to create sub-microliter hydrophobic wells surrounding each sensor to eliminate cross contamination during immobilization of capture antibodies. High-throughput analyses were realized using eight 32-sensor immunoarrays connected to the miniaturized 8-port manifold, allowing 256 measurements in <1 h. This system was used to determine prostate cancer biomarker proteins prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), interleukin-6 (IL-6), and platelet factor-4 (PF-4) in serum. Clinically relevant detection limits (0.05 to 2 pg mL-1) and 5-decade dynamic ranges (sub pg mL-1 to well above ng mL-1) were achieved for these proteins utilizing precapture of analyte proteins on magnetic nanoparticles decorated with enzyme labels and antibodies.