Microfluidic Array Research Articles

Crack-Enhanced Microfluidic Stretchable E-Skin Sensor.

We reported the development of a transparent stretchable crack-enhanced microfluidic capacitive sensor array for use in E-skin applications. The microfluidic sensor was fabricated through a simple lamination process involving two silver nanowire (AgNW)-embedded rubbery microfluidic channels arranged in a crisscross fashion. The sensing performance was optimized by testing a variety of sensing liquids injected into the channels. External mechanical stimuli applied to the sensor induced the liquid to penetrate the deformed microcracks on the rubber channel surface. The increased interfacial contact area between the liquid and the nanowire electrodes increased the capacitance of the sensor. The device sensitivity was strongly related to both the initial fluid interface between the liquid and crack wall and the change in the contact length of the liquid and crack wall, which were simulated using the finite element method. The microfluidic sensor was shown to detect a wide range of pressures, 0.1-140 kPa. Ordinary human motions, including substantial as well as slight muscle movements, could be successively detected, and 2D color mappings of simultaneous external load sensing were collected. Our simple method of fabricating the microfluidic channels and the application of these channels to stretchable e-skin sensors offers an excellent sensing platform that is highly compatible with emerging medical and electronic applications.

Combined microRNA and mRNA microfluidic TaqMan array cards for the diagnosis of malignancy of multiple types of pancreatico-biliary tumors in fine-needle aspiration material.

Pancreatic ductal adenocarcinoma (PDAC) continues to carry the lowest survival rates among all solid tumors. A marked resistance against available therapies, late clinical presentation and insufficient means for early diagnosis contribute to the dismal prognosis. Novel biomarkers are thus required to aid treatment decisions and improve patient outcomes.We describe here a multi-omics molecular platform that allows for the first time to simultaneously analyze miRNA and mRNA expression patterns from minimal amounts of biopsy material on a single microfluidic TaqMan Array card. Expression profiles were generated from 113 prospectively collected fine needle aspiration biopsies (FNAB) from patients undergoing surgery for suspect masses in the pancreas. Molecular classifiers were constructed using support vector machines, and rigorously evaluated for diagnostic performance using 10×10fold cross validation. The final combined miRNA/mRNA classifier demonstrated a sensitivity of 91.7%, a specificity of 94.5%, and an overall diagnostic accuracy of 93.0% for the differentiation between PDAC and benign pancreatic masses, clearly outperfoming miRNA-only classifiers. The classification algorithm also performed very well in the diagnosis of other types of solid tumors (acinar cell carcinomas, ampullary cancer and distal bile duct carcinomas), but was less suited for the diagnostic analysis of cystic lesions.We thus demonstrate that simultaneous analysis of miRNA and mRNA biomarkers from FNAB samples using multi-omics TaqMan Array cards is suitable to differentiate suspect solid pancreatic masses with high precision.

Dynamically Gas‐Phase Switchable Super(de)wetting States by Reversible Amphiphilic Functionalization: A Powerful Approach for Smart Fluid Gating Membranes

AbstractIn nature, cellular membranes perform critical functions such as endocytosis and exocytosis through smart fluid gating processes mediated by nonspecific amphiphilic interactions. Despite considerable progress, artificial fluid gating membranes still rely on laborious stimuli‐responsive mechanisms and triggering systems. In this study, a room temperature gas‐phase approach is presented for dynamically switching a porous material from a superhydrophobic to a superhydrophilic wetting state and back. This is realized by the reversible attachment of bipolar amphiphiles, which promote surface wetting. Application of this reversible amphiphilic functionalization to an impermeable nanofibrous membrane induces a temporary state of superhydrophilicity resulting in its pressure‐less permeation. This mechanism allows for rapid smart fluid gating processes that can be triggered at room temperature by variations in the environment of the membrane. Owing to the universal adsorption of volatile amphiphiles on surfaces, this approach is applicable to a broad range of materials and geometries enabling facile fabrication of valve‐less flow systems, fluid‐erasable microfluidic arrays, and sophisticated microfluidic designs.

On-Chip Glass Microspherical Shell Whispering Gallery Mode Resonators

Arrays of on-chip spherical glass shells of hundreds of micrometers in diameter with ultra-smooth surfaces and sub-micrometer wall thicknesses have been fabricated and have been shown to sustain optical resonance modes with high Q-factors of greater than 50 million. The resonators exhibit temperature sensitivity of −1.8 GHz K−1 and can be configured as ultra-high sensitivity thermal sensors for a broad range of applications. By virtue of the geometry’s strong light-matter interaction, the inner surface provides an excellent on-chip sensing platform that truly opens up the possibility for reproducible, chip scale, ultra-high sensitivity microfluidic sensor arrays. As a proof of concept we demonstrate the sensitivity of the resonance frequency as water is filled inside the microspherical shell and is allowed to evaporate. By COMSOL modeling, the dependence of this interaction on glass shell thickness is elucidated and the experimentally measured sensitivities for two different shell thicknesses are explained.

A multiplex microfluidic loop-mediated isothermal amplification array for detection of malaria-related parasites and vectors

Malaria infection poses a great threaten to public health even nowadays. The conventional diagnosis tools of malaria parasites and vectors require systematic training for the observers accompanied by the low throughput. In this study, a new detection system, i.e., multiplex microfluidic loop-mediated isothermal amplification (mμLAMP) array, was developed to provide a convenient, rapid and economical detection system for malaria diagnosis. A microfluidic-based detection chip was designed and developed, targeting the conserved gene of four Anopheles and two Plasmodium species responsible for most of the malaria cases occurred in China. The DNA preparation of Anopheles and Plasmodium samples was realized by using a newly-developed DNA extraction method. For this mμLAMP array system, the detection limit was determined to be 1pg of targeting DNA with high sensitivity (>95%) and specificity (100%). Further, the accuracy of such mμLAMP analysis was evaluated by the analysis of 48 Anopheles mosquito samples, of which 30 were termed to be target Anopheles, displaying high consistency with that by morphological analysis. In conclusion, the mμLAMP detection system was proved to be a visible, sensitive, specific and high-throughput diagnostic tool. Considering the portable manipulation of such detection system, our studies shed light on its potential application of malaria surveillance on the spot.

Orthogonal Screening of Anticancer Drugs Using an Open-Access Microfluidic Tissue Array System.

Screening for potential drug combinations presents significant challenges to the current microfluidic cell culture systems, due to the requirement of flexibility in liquid handling. To overcome this limitation, we present here an open-access microfluidic tissue array system specifically designed for drug combination screening. The microfluidic chip features a key structure in which a nanoporous membrane is sandwiched by a cell culture chamber array layer and a corresponding media reservoir array layer. The microfluidic approach takes advantage of the characteristics of the nanoporous membrane: on one side, this membrane permits the flow of air but not liquid, thus acting as a flow-stop valve to enable automatic cell distribution; on the other side, it allows diffusion-based media exchange and thus mimics the endothelial layer. In synergy with a liquid-transferring platform, the open-access microfluidic system enables complex multistep operations involving long-term cell culture, medium exchange, multistep drug treatment, and cell-viability testing. By using the microfluidic protocol, a 10 × 10 tissue array was constructed in 90 s, followed by schedule-dependent drug testing. Morphological and immunohistochemical assays indicated that the resultant tumor tissue was faithful to that in vivo. Drug-testing assays showed that the incorporation of the nanoporous membrane further decreased killing efficacy of the anticancer agents, indicating its function as an endothelial layer. Robustness of the microfluidic system was demonstrated by implementing a three-factor, three-level orthogonal screening of anticancer drug combinations, with which 67% of the testing (9 vs. 27) was saved. Experimental results demonstrated that the microfluidic tissue system presented herein is flexible and easy-to-use, thus providing an ideal tool for performing complex multistep cell assays with high efficiencies.

Resolving Species Level Changes in a Representative Soil Bacterial Community Using Microfluidic Quantitative PCR.

Rapid advances in genome sequencing technologies enable determination of relative bacterial abundances and community composition, yet, changes at the species level remain difficult to detect despite importance for certain ecological inferences. We present a method for extraction and direct quantification of species composition of a predefined multispecies bacterial community using microfluidic-based quantitative real-time PCR (qPCR). We employ a nested PCR approach based on universal 16S rRNA gene pre-amplification followed by detection and quantification of absolute abundance of bacterial species using microfluidic array of parallel singleplex qPCR reactions. Present microfluidic qPCR supports 2,304 simultaneous reactions on a single chip, while automatic distribution of samples and reactants minimizes pipetting errors and technical variations. The utility of the method is illustrated using a synthetic soil bacterial community grown in two contrasting environments – sand microcosms and batch cultures. The protocol entails extraction of total nucleic acid, preparation of genomic DNA, and steps for qPCR assessment of bacterial community composition. This method provides specific and sensitive quantification of bacterial species requiring only 2 ng of community DNA. Optimized extraction protocol and preamplification step allow for rapid, quantitative, and simultaneous detection of candidate species with high throughput. The proposed method offers a simple and accurate alternative to present sequencing methods especially when absolute values of species abundance are required. Quantification of changes at the species level contributes to the mechanistic understanding of the roles of particular species in a bacterial community functioning.

Optimizing Multiplexed Detections of Diabetes Antibodies via Quantitative Microfluidic Droplet Array.

Sensitive, single volume detections of multiple diabetes antibodies can provide immunoprofiling and early screening of at-risk patients. To advance the state-of-the-art suspension assays for diabetes antibodies, porous hydrogel droplets are leveraged in microfluidic serpentine arrays to enhance reagent transport. This spatially multiplexed assay is applied to the detection of antibodies against insulin, glutamic acid decarboxylase, and insulinoma-associated protein 2. Optimization of assay protocol results in a shortened assay time of 2 h, with better than 20 pg mL Supporting Information detection limits across all three antibodies. Specificity and cross-reactivity tests show negligible background, nonspecific antibody-antigen, and nonspecific antibody-antibody bindings. Multiplexed detections are able to measure within 15% of target concentrations from low to high ranges. The technique enables quantifications of as little as 8000 molecules in each 500 µm droplet in a single volume, multiplexed assay format, a breakthrough necessary for the adoption of diabetes panels for clinical screening and monitoring in the future.

Three-Dimensional Cell Culture and Drug Testing in a Microfluidic Sidewall-Attached Droplet Array.

Three-dimensional (3D) cell culture provides an effective way over conventional two-dimensional (2D) monolayer culture to more closely imitate the complex cellular organization, heterogeneity, and interactions as well as tissue microenvironments in vivo. Here we present a novel droplet-based 3D cell culture method by using droplet array attached on the sidewall of a PDMS piece. Such an arrangement not only avoids cells from adhering on the chip surface for achieving 3D cell culture in nanoliter-scale droplets, but also facilitates performing multiple operations to cells in droplets, including cell suspension droplet generation, drug treatment, and cell staining with a capillary-based liquid handling system, as well as in situ observation and direct scanning with a confocal laser scanning microscope. We optimized the system by studying the effects of various conditions to cell culture including droplet volume, cell density and fabrication methods of the PDMS pieces. We have applied this system in the 3D culture of HepG2 cells and the stimulation testing of an anticancer drug, doxorubicin, to 3D cell spheroids.

Assessing SNP genotyping of noninvasively collected wildlife samples using microfluidic arrays

Noninvasively collected samples are a common source of DNA in wildlife genetic studies. Currently, single nucleotide polymorphism (SNP) genotyping using microfluidic arrays is emerging as an easy-to-use and cost-effective methodology. Here we assessed the performance of microfluidic SNP arrays in genotyping noninvasive samples from grey wolves, European wildcats and brown bears, and we compared results with traditional microsatellite genotyping. We successfully SNP-genotyped 87%, 80% and 97% of the wolf, cat and bear samples, respectively. Genotype recovery was higher based on SNPs, while both marker types identified the same individuals and provided almost identical estimates of pairwise differentiation. We found that samples for which all SNP loci were scored had no disagreements across the three replicates (except one locus in a wolf sample). Thus, we argue that call rate (amplification success) can be used as a proxy for genotype quality, allowing the reduction of replication effort when call rate is high. Furthermore, we used cycle threshold values of real-time PCR to guide the choice of protocols for SNP amplification. Finally, we provide general guidelines for successful SNP genotyping of degraded DNA using microfluidic technology.

Programmable Static Droplet Array for the Analysis of Cell-Cell Communication in a Confined Microenvironment.

Direct cell-cell communication can occur through various chemical and mechanical signals. However, available cell culture systems lack single-cell resolution and are often limited by sensitivity and accuracy. In this study, we present an accurate, efficient and controllable microfluidic device that can be used for in situ monitoring of natural cell-cell contact and signaling processes in a confined microenvironment. This innovative static droplet array (SDA) enables highly efficient trapping, encapsulation, arraying, storage, and incubation of defined cell populations. For proof-of-principle experiments, we monitored the response of budding yeast to peptide mating pheromones, as it is one of the best understood examples of eukaryotic cell-cell communication. Specifically, we measured the yeast response to varying concentration of synthetic MATα-type mating factor, as well as varying the cell number ratio of MATα and MATa in a confined space. We found clear morphological and doubling-time changes during the mating reaction with a significantly higher accuracy than conventional methods. Further, phenotypic analysis of data generated with the microfluidic static droplet array allowed distinguishing the function of genes in yeast mutants defective for different aspects of pheromone signaling. Taken together, the microfluidic platform provides a valuable research tool to study cell-cell communication and signaling in a controlled microenvironment with the sensitivity and accuracy required for screening and long-term phenotypic analysis.

On-chip cell labelling and washing by capture and release using microfluidic trap arrays.

Flow cytometry analysis requires a large amount of isolated, labelled, and purified cells for accurate results. To address the demand for a large quantity of cells prepared in a timely manner, we describe a novel microfluidic trap structure array for on-chip cell labelling, such as intracellular and extracellular labelling, and subsequent washing and release of cells. Each device contains [Formula: see text] trap structures, which made the preparation of large numbers of cells [Formula: see text] possible. The structure has a streamlined shape, which minimizes clogging of cells in capture and release steps. The trap structure arrays are built and tested using leukocytes, with different load flow speeds, incubation times, and release flow speeds. ∼85% of cells are captured independent of the input flow speed. The release efficiency depends on the incubation time, with over ∼80% of captured cells released for up to 20 min incubation, and on-chip labelling and washing with STYO13 are demonstrated. Qualitative models are developed as guidance for designing the proposed trap structure and to explain the increased performance over previous approaches.

Field-Deployable Wireless Sensor Networks for Mapping Inorganic N-Containing Species in Soil

Conventional practice of applying fertilizer and irrigation are empirical, often resulting in negative consequences to the environment and human welfare due to the insufficient or excess usage of fertilizer and water. Hence there is a persistent need for the knowledge to assist farmers in determining the quantity and timing of fertilizer and water application to maximize crop yield, minimize waste, and reduce cost. In this study, we aim to develop a highly sensitive radio frequency microfluidic coplanar waveguide array (MCWA) based sensor that can measure N-containing species down to the 3 picomolar by detecting small changes in permittivity of the sample solution upon analyte binding. Based on the single senor unit, we will develop an integrated MCWA sensor array by constructing a series of electrical and microfluidic circuits with each of the sensor units targeting specific analyte species for simultaneous measurement of different N-containing species simultaneously. The MCWA sensor array will be further integrated into a field-deployable soil probe and used to demonstrate the ability of mapping nitrate, ammonium, pH and soil moisture content on the farm using a network of sensor probes spread over a large area. The data from the sensor networks can be used to determine when and how much fertilizer and water should be applied, enabling just-in-time and just-enough geospatial applications of fertilizer and irrigation water, which would contribute to the food-energy-water nexus by increasing crop yield and, at the same time, reducing usage of water and fertilizer.

Microfluidic single-cell array platform enabling week-scale clonal expansion under chemical/electrical stimuli

Single-cell culture represents the most straightforward method for investigating cellular heterogeneity. In this paper, we present a novel microfluidic platform that can individually array and culture hundreds of cells under chemical and electrical stimuli for week-scale characterization. Single cells can be deterministically and gently captured in a microchamber array on the proposed platform. The size of the microchamber can be adjusted to fit different cell culture times, and this characteristic enables remarkable scalability. Transparent indium tin oxide microelectrodes were integrated with the single-cell array platform for on-chip electrical stimuli. The platform exhibited nearly 90% single-cell efficiency and facilitated week-scale clonal expansion of different types of single cells. Chemical and electrical stimuli affected proliferation and differentiation of MC 3T3-E1 cells were examined on the chip prototype that contained 416 (32 rows × 13 columns) microchambers, and each microchamber had 1 mm diameter. By tracking clonal expansion of cells under chemical/electrical stimuli for relatively long periods, the proposed platform can facilitate the screening of the cell subpopulation with a favorable growth phenotype for drug testing and cell therapy.

Ehrlichia chaffeensis TRP120 Moonlights as a HECT E3 Ligase Involved in Self- and Host Ubiquitination To Influence Protein Interactions and Stability for Intracellular Survival.

Ehrlichia chaffeensis secretes tandem repeat protein (TRP) effectors that are involved in a diverse array of host cell interactions, some of which directly activate cell signaling pathways and reprogram host gene transcription to promote survival in the mononuclear phagocyte. However, the molecular details of these effector-host interactions and roles in pathobiology are incompletely understood. In this study, we determined that the E. chaffeensis effector TRP120 is posttranslationally modified by ubiquitin (Ub) and that ubiquitination occurs through intrinsic and host-mediated HECT ligase activity. A functional HECT E3 ligase domain with a conserved catalytic site was identified in the C-terminal region of TRP120, and TRP120 autoubiquitination occurred in vitro in the presence of host UbcH5b/c E2 enzymes. TRP120 ubiquitination sites were mapped using a high-density microfluidic peptide array and confirmed by ectopic expression of TRP120 lysine mutants in cells. Moreover, we determined that the HECT E3 ubiquitin ligase, Nedd4L, interacts with TRP120 during infection and also mediates TRP120 ubiquitination. Nedd4L knockdown resulted in the reduction of TRP120-Ub, decreased ehrlichial infection, and reduced recruitment of a known TRP120-interacting host protein, PCGF5, to ehrlichial inclusions. TRP120-mediated PCGF5 polyubiquitination was associated with a reduction in PCGF5 levels. Inhibition of ubiquitination with small molecules also significantly decreased ehrlichial infection, indicating that the Ub pathway is critical for ehrlichial intracellular replication and survival. The current study identified a novel E. chaffeensis ubiquitin ligase and revealed an important role for the ubiquitin pathway in effector-host interactions and pathogen-mediated host protein stability in order to promote intracellular survival.

Nanoscale Hydrodynamic Film for Diffusive Mass Transport Control in Compartmentalized Microfluidic Chambers.

A compartmentalized microfluidic chamber array that offers not only separate cell culture environments but also independent control of the diffusion of small molecules provides an extremely useful platform for cell cultivations and versatile cellular assays. However, it is challenging to incorporate both cell compartmentalization and active diffusion control in real-time and precise manners. Here, we present a novel nanoscale hydrodynamic film (NHF) that is formed between a solid substrate and a polydimethylsiloxane (PDMS) surface. The thickness of the NHF can be adjusted by varying the pressure applied between them so that the mass transfer through the NHF can also be controlled. These novel phenomena are characterized and applied to develop a compartmentalized microchamber array with diffusion-tunable and solution-switchable chemostat-like versatile bacterial assays. The NHF-based compartmentalization technique is ideal for not only continuous bacterial cultivation by consistently refreshing various nutrient sources but also various diffusion-based microbial assays such as chemical induction of synthetically engineered bacterial cells and selective growth of a specific bacterial strain with respect to chemical environments. In addition, we show that tight compartmentalization protects cells in the chambers, while biofilm formation and nutrient contamination are eliminated by loading a lysis buffer, which typically hinders long-term continuous cultures and accurate microbial assays on a chip. Therefore, we ensure that the NHF-based compartmentalization platform proposed in this work will facilitate not only fundamental studies in microbiology but also various practical applications of microbes for production of valuable metabolites and byproducts in a high-throughput and highly efficient format.

Fully disposable microfluidic electrochemical device for detection of estrogen receptor alpha breast cancer biomarker

A novel fully disposable microfluidic electrochemical array device (µFED) was developed and successfully applied for detection of the biomarker estrogen receptor alpha (ERα). The µFED was constructed using low-cost materials and an inexpensive home cutter printer enabled the manufacture of dozens of µFEDs in less than 2h, at a cost of less than US$ 0.20 in material per device. The µFED incorporates counter and reference electrodes and eight carbon-based working electrodes, which were modified with DNA sequences known as estrogen response elements (DNA-ERE), where ERα binds specifically. Paramagnetic particles heavily decorated with anti-ERα antibody and horseradish peroxidase (MP-Ab-HRP) were used to efficiently capture ERα from the sample solution. The ERα-MP-Ab-HRP bioconjugate formed was injected into the µFED and incubated with the DNA-ERE-modified electrodes, followed by amperometric detection with application of −0.2V vs. Ag|AgCl while a mixture of H2O2 and hydroquinone was injected into the microfluidic device. An ultralow limit of detection of 10.0 fg mL−1 was obtained with the proposed method. The performance of the assay, in terms of sensitivity and reproducibility, was studied using undiluted calf serum, and excellent recoveries in the range of 94.7–108% were achieved for the detection of ERα in MCF-7 cell lysate. The µFED system can be easily constructed and applied for multiplex biomarker detection, making the device an excellent cost-effective alternative for cancer diagnosis, especially in developing countries.

Micro-Viscometer for Measuring Shear-Varying Blood Viscosity over a Wide-Ranging Shear Rate.

In this study, a micro-viscometer is developed for measuring shear-varying blood viscosity over a wide-ranging shear rate. The micro-viscometer consists of 10 microfluidic channel arrays, each of which has a different micro-channel width. The proposed design enables the retrieval of 10 different shear rates from a single flow rate, thereby enabling the measurement of shear-varying blood viscosity with a fixed flow rate condition. For this purpose, an optimal design that guarantees accurate viscosity measurement is selected from a parametric study. The functionality of the micro-viscometer is verified by both numerical and experimental studies. The proposed micro-viscometer shows 6.8% (numerical) and 5.3% (experimental) in relative error when compared to the result from a standard rotational viscometer. Moreover, a reliability test is performed by repeated measurement (N = 7), and the result shows 2.69 ± 2.19% for the mean relative error. Accurate viscosity measurements are performed on blood samples with variations in the hematocrit (35%, 45%, and 55%), which significantly influences blood viscosity. Since the blood viscosity correlated with various physical parameters of the blood, the micro-viscometer is anticipated to be a significant advancement for realization of blood on a chip.

Ultrasensitive Detection of Cancer Biomarkers in the Clinic by Use of a Nanostructured Microfluidic Array

Ultrasensitive Detection of Cancer Biomarkers in the Clinic by Use of a Nanostructured Microfluidic Array

Large-scale microfluidic gradient arrays reveal axon guidance behaviors in hippocampal neurons

High-throughput quantitative approaches to study axon growth behaviors have remained a challenge. We have developed a 1024-chamber microfluidic gradient generator array that enables large-scale investigations of axon guidance and growth dynamics from individual primary mammalian neurons, which are exposed to gradients of diffusible molecules. Our microfluidic method (a) generates statistically rich data sets, (b) produces a stable, reproducible gradient with negligible shear stresses on the culture surface, (c) is amenable to the long-term culture of primary neurons without any unconventional protocol, and (d) eliminates the confounding influence of cell-secreted factors. Using this platform, we demonstrate that hippocampal axon guidance in response to a netrin-1 gradient is concentration-dependent—attractive at higher concentrations and repulsive at lower concentrations. We also show that the turning of the growth cone depends on the angle of incidence of the gradient. Our study highlights the potential of microfluidic devices in producing large amounts of data from morphogen and chemokine gradients that play essential roles not only in axonal navigation but also in stem cell differentiation, cell migration, and immune response.