On-chip electrochemical detection of CdS quantum dots using normal and multiple recycling flow through modes

A flexible hybrid polydimethylsiloxane (PDMS)-polycarbonate (PC) microfluidic chip with integrated screen printed electrodes (SPE) was fabricated and applied for electrochemical quantum dots (QDs) detection. The developed device combines the advantages of flexible microfluidic chips, such as their low cost, the possibility to be disposable and amenable to mass production, with the advantages of electrochemistry for its facility of integration and the possibility to miniaturize the analytical device. Due to the interest in biosensing applications in general and particularly the great demand for labelling alternatives in affinity biosensors, the electrochemistry of cadmium sulfide quantum dots (CdS QDs) is evaluated. Square wave anodic stripping voltammetry (SWASV) is the technique used due to its sensitivity and low detection limits that can be achieved. The electrochemical as well as the microfluidic parameters of the developed system are optimized. The detection of CdS QDs in the range between 50 to 8000 ng mL(-1) with a sensitivity of 0.0009 μA/(ng mL(-1)) has been achieved. In addition to the single in-chip flow through measurements, the design of a recirculation system with the aim of achieving lower detection limits using reduced volumes (25 μL) of sample was proposed as a proof-of-concept.     

Endothelial cell polarization and chemotaxis in a microfluidic device

The directed migration of endothelial cells is an early and critical step in angiogenesis, or new blood vessel formation. In this study, the polarization and chemotaxis of human umbilical vein endothelial cells (HUVEC) in response to quantified gradients of vascular endothelial growth factor (VEGF) were examined. To accomplish this, a microfluidic device was designed and fabricated to generate stable concentration gradients of biomolecules in a cell culture chamber while minimizing the fluid shear stress experienced by the cells. Finite element simulation of the device geometry produced excellent agreement with the observed VEGF concentration distribution, which was found to be stable across multiple hours. This device is expected to have wide applicability in the study of shear-sensitive cells such as HUVEC and non-adherent cell types as well as in the study of migration through three-dimensional matrices. HUVEC were observed to chemotax towards higher VEGF concentrations across the entire range of concentrations studied (18-32 ng mL(-1)) when the concentration gradient was 14 ng mL(-1) mm(-1). In contrast, shallow gradients (2 ng mL(-1) mm(-1)) across the same concentration range were unable to induce HUVEC chemotaxis. Furthermore, while all HUVEC exposed to elevated VEGF levels (both in steep and shallow gradients) displayed an increased number of filopodia, only chemotaxing HUVEC displayed an asymmetric distribution of filopodia, with enhanced numbers of protrusions present along the leading edge. These results suggest a two-part requirement to induce VEGF chemotaxis: the VEGF absolute concentration enhances the total number of filopodia extended while the VEGF gradient steepness induces filopodia localization, cell polarization, and subsequent directed migration.     

Micropillar‐based microfluidic device to regulate neurite networks of uniform‐sized neurospheres

The inability of neurons to undergo mitosis renders damage to the central or peripheral nervous system. Neural stem cell therapy could provide a path for treating the neurodegenerative diseases. However, reliable and simple tools for the developing and testing neural stem cell therapy are still required. Here, we show the development of a micropillar‐based microfluidic device to trap the uniform‐sized neurospheres. The neurospheres trapped within micropillar arrays were largely differentiated into neuronal cells, and their neurite networks were observed in the microfluidic device. Compared to conventional cultures on glass slides, the neurite networks generated with this method have a higher reproducibility. Furthermore, we demonstrated the effect of thapsigargin on the neurite networks in the microfluidic device, demonstrating that neural networks exposed to thapsigargin were largely diminished and disconnected from each other. Therefore, this micropillar‐based microfluidic device could be a potential tool for screening of neurotoxins.     

Epithelial‐to‐mesenchymal transition of human lung alveolar epithelial cells in a microfluidic gradient device

Epithelial‐to‐mesenchymal transition (EMT), a process in which epithelial cells undergo phenotypic transitions to fibrotic cells, is induced by stimulants including transforming growth factor‐beta1 (TGF‐β1). In the present study, we developed a microfluidic gradient device to reproduce EMT in A549 human lung alveolar epithelial cells in response to TGF‐β1 gradients. The device was directly mounted on the cells that had grown in cell culture plates and produced a stable concentration gradient of TGF‐β1 with negligible shear stress, thereby providing a favorable environment for the anchorage‐dependent cells. A549 cells elongated with the characteristic spindle‐shaped morphological changes with upregulation of alpha‐smooth muscle actin, a mesenchyme marker, in a gradient‐dependent manner, suggestive of EMT progression. We observed that at higher TGF‐β1 concentrations ranging from 5 to 10 ng/mL, the cultures in the microfluidic device allowed to quantitatively pick up subtle differences in the EMT cellular response as compared with plate cultures. These results suggest that the microfluidic gradient device would accurately determine the optimal concentrations of TGF‐β1, given that epithelial cells of different tissue origins greatly vary their responses to TGF‐β1. Therefore, this microfluidic device could be a powerful tool to monitor EMT induced by a variety of environmental stresses including cigarette smoke with high sensitivity.     

Magnetic force-based multiplexed immunoassay using superparamagnetic nanoparticles in microfluidic channel

This paper describes a novel microfluidic immunoassay utilizing binding of superparamagnetic nanoparticles to beads and deflection of these beads in a magnetic field as the signal for measuring the presence of analyte. The superparamagnetic 50 nm nanoparticles and fluorescent 1 microm polystyrene beads are immobilized with specific antibodies. When target analytes react with the polystyrene beads and superparamagnetic nanoparticles simultaneously, the superparamagnetic nanoparticles can be attached onto the microbeads by the antigen-antibody complex. In the poly(dimethylsiloxane)(PDMS) microfluidic channel, only the microbeads conjugated with superparamagnetic nanoparticles by analytes consequently move to the high gradient magnetic fields under the specific applied magnetic field. In this study, the magnetic force-based microfluidic immunoassay is successfully applied to detect the rabbit IgG and mouse IgG as model analytes. The lowest concentration of rabbit IgG and mouse IgG measured over the background is 244 pg mL(-1) and 15.6 ng mL(-1), respectively. The velocities of microbeads conjugated with superparamagnetic nanoparticles are demonstrated by magnetic field gradients in microfluidic channels and compared with the calculated magnetic field gradients. Moreover, dual analyte detection in a single reaction is also performed by the fluorescent encoded microbeads in the microfluidic device. Detection range and lower detection limit can be controlled by the microbeads concentration and the higher magnetic field gradient.     

Rapid ultraviolet monitoring of multiple psychotropic drugs with a renewable microfluidic device

A rapid method for sensitive ultraviolet detection of multiple psychotropic drugs in human plasma was developed on a low-cost and expediently fabricated hybrid microfluidic device. The device was composed of one fused-silica capillary with a sampling fracture, a poly(methyl methacrylate) board with four reservoirs, and a printed circuit board. At the optimal separation and detection conditions, the baseline separation of three kinds of psychotropic drugs including barbiturates (phenobarbital and barbital), benzodiazepines (nitrazepam, clonazepam, chlordiazepoxide, alprazolam and diazepam) and tricyclic antidepressant drugs (amitriptyline) was achieved within 200 s with separation efficiency up to 3.80 × 10(5) plates m(-1). The linear ranges for ultraviolet detection were from 2.0 to 1000.0 μg mL(-1) for chlordiazepoxide and 1.0 to 1000.0 μg mL(-1) for other seven drugs. Combining with solid-phase extraction, this novel protocol could successfully be used to screen naturally existing psychotropic drugs in a known human plasma sample. The minimum detectable concentration was down to 27 ng mL(-1) for phenobarbital spiked in plasma. This work provided a promising way to initially screen different psychotropic drugs with high resolution, rapid separation and low-cost.     

Rapid detection of algal toxins by microfluidic immunoassay

Herein we report fabricating a microfluidic device to monitor harmful algal blooming (HAB). The heterogeneous immuno-enzyme assay was integrated into a self-designed microfluidic chip for rapid and automatic analysis of algal toxins. The device was made from polydimethylsiloxane (PDMS) and was assembled with a home-made control system. The performance of the system was demonstrated by the detection of microcystin, saxitoxin and cylindrospermopsin, the major cyanotoxins. In one single microfluidic chip, multiple samples were controlled and analysed in a parallel manner. Under the optimal conditions, the linear range and the limit of detection of microcystins were 0-5.0 ng mL(-1) and 0.02 ng mL(-1) respectively. The total analysis time was less than 25 min. The designed device was highly automatic, more efficient and economic compared to conventional techniques.     

A microfluidic device for the automated derivatization of free fatty acids to fatty acid methyl esters

Free fatty acid (FFA) compositions are examined in feedstock for biodiesel production, as source-specific markers in soil, and because of their role in cellular signaling. However, sample preparation of FFAs for gas chromatography-mass spectrometry (GC-MS) analysis can be time and labor intensive. Therefore, to increase sample preparation throughput, a glass microfluidic device was developed to automate derivatization of FFAs to fatty acid methyl esters (FAMEs). FFAs were delivered to one input of the device and methanolic-HCl was delivered to a second input. FAME products were produced as the reagents traversed a 29 μL reaction channel held at 55 °C. A Design of Experiment protocol was used to determine the combination of derivatization time (T(der)) and ratio of methanolic-HCl:FFA (R(der)) that maximized the derivatization efficiencies of tridecanoic acid and stearic acid to their methyl ester forms. The combination of T(der) = 0.8 min and R(der) = 4.9 that produced optimal derivatization conditions for both FFAs within a 5 min total sample preparation time was determined. This combination of T(der) and R(der) was used to derivatize 12 FFAs with a range of derivatization efficiencies from 18% to 93% with efficiencies of 61% for tridecanoic acid and 84% for stearic acid. As compared to a conventional macroscale derivatization of FFA to FAME, the microfluidic device decreased the volume of methanolic-HCl and FFA by 20- and 1300-fold, respectively. The developed microfluidic device can be used for automated preparation of FAMEs to analyze the FFA compositions of volume-limited samples.     

Microfluidic nanoplasmonic-enabled device for multiplex DNA detection

We describe a rapid, quantitative, multiplex, self-labelled, and real-time DNA biosensor employing Ag nanoparticle-bound DNA hairpin probes immobilized in a microfluidic channel. Capture of complementary target DNAs by the microarrayed DNA hairpin probes results in a positive fluorescence signal via a conformational change of the probe molecules, signalling the presence of target DNAs. The device's capability for quantitative analyses was evaluated and a detection time as low as 6 min (with a target flow rate of 0.5 μl min(-1)) was sufficient to generate significant detection signals. This detection time translates to merely 3 μl of target solution consumption. An unoptimized sensitivity of 500 pM was demonstrated for this device.     

Rapid point-of-care concentration of bacteria in a disposable microfluidic device using meniscus dragging effect

We report a low cost, disposable polymer microfluidic sample preparation device to perform rapid concentration of bacteria from liquid samples using enhanced evaporation targeted at downstream detection using surface enhanced Raman spectroscopy (SERS). The device is composed of a poly(dimethylsiloxane) (PDMS) liquid sample flow layer, a reusable metal airflow layer, and a porous PTFE (Teflon?) membrane sandwiched in between the liquid and air layers. The concentration capacity of the device was successfully demonstrated with fluorescently tagged Escherichia coli (E. coli). The recovery concentration was above 85% for all initial concentrations lower than 1 × 10(4) CFU mL(-1). In the lowest initial concentration cases, 100 μL initial volumes of bacteria solution at 100 CFU mL(-1) were concentrated into 500 nL droplets with greater than 90% efficiency in 15 min. Subsequent tests with SERS on clinically relevant Methicillin-Sensitive Staphylococcus aureus (MSSA) after concentration in this device proved more than 100-fold enhancement in SERS signal intensity compared to the signal obtained from the unconcentrated sample. The concentration device is straightforward to design and use, and as such could be used in conjunction with a number of detection technologies.     

集成核酸提取的实时荧光PCR微全分析系统

集成核酸提取的实时荧光PCR微全分析系统将核酸提取、PCR扩增与实时荧光检测进行整合,在同一块微流控芯片上实现了核酸分析过程的全自动和全封闭,具有试剂用量少、分析速度快、操作简便等优点。本研究采用微机械加工技术制作集成核酸提取微流控芯片的阳极模,使用组合模具法和注塑法制作具有3D通道的PDMS基片,与玻璃基底通过等离子体键合封装成集成核酸提取芯片。构建了由微流体速度可调节(0~10 mL/min)的驱动控制装置、温控精度可达0.1℃的TEC温控平台、CCD检测功能模块等组成的微全分析系统。以人类血液裂解液为样品,采用硅胶膜进行芯片上核酸提取。系统根据设置好的时序自动执行,以2 mL/min的流体驱动速度完成20μL裂解液上样、清洗;以1 mL/min的流体驱动速度完成DNA洗脱,抽取PCR试剂与之混合注入到反应腔。提取的基因组DNA以链上内参基因GAPDH为检测对象,并以传统手工提取为对照,在该系统平台上进行PCR扩增和熔解曲线分析实验。片上PCR扩增结果显示,扩增曲线明显,Ct值分别为25.3和26.9。扩增产物进行熔解曲线分析得到的熔解温度一致,均为89.9℃。结果表明,此系统能够自动化、全封闭的在微流控芯片上完成核酸提取、PCR扩增与实时定量分析。     

Microfluidic hydrogel layers with multiple gradients to stimulate and perfuse three-dimensional neuronal cell cultures

We present a simple and easy to handle PDMS microfluidic device for neuronal cell culture studies in three-dimensional hydrogel scaffolds. The hydrogel is structured in parallel layers to reconstruct cell layers close to the natural environment. Dissociated cortical neurons of embryonic rats have been cultured in 0.5% w/v agarose including 0.2% w/v alginate. The cells formed neurite networks through neighboring cell free hydrogel layers. The cell culture showed neurite outgrowth in the microfluidic channel over more than seven days in vitro without perfusion. Culturing neurons in hydrogel layers surrounded by a liquid phase containing culture medium resulted in denser neuronal networks.     

Mucin (MUC5AC) expression by lung epithelial cells cultured in a microfluidic gradient device

We have developed a microfluidic gradient device for controlling mucin gene expression of NCI-H292 epithelial cells derived from lung tissues. We hypothesized that gradient profiles would control mucin gene expression of lung epithelial cells. However, it was not possible to generate various stable gradient profiles using conventional culture methods. To address this limitation, we used a microfluidic gradient device to create various gradient profiles (i.e. non-linear, linear, and flat) in a temporal and spatial manner. NCI-H292 lung epithelial cells were exposed to concentration gradients of epidermal growth factor in a microfluidic gradient device with continuous medium perfusion. We demonstrated an effect of gradient profiles on mucin expression of lung epithelial cells cultured in the microfluidic gradient device. It was revealed that NCI-H292 lung epithelial cells exposed to the flat gradient profile of the epidermal growth factor exhibited high expression of mucin as compared with cells exposed to non-linear and linear gradient profiles. Therefore, this microfluidic gradient device could be a potentially useful tool for regulating the mucin expression of lung epithelial cells exposed to chemokine gradient profiles.     

Characterization of collagen thin films for von Willebrand factor binding and platelet adhesion

Von Willebrand factor (VWF) binding and platelet adhesion to subendothelial collagens are initial events in thrombus formation at sites of vascular injury. These events are often studied in vitro using flow assays designed to mimic vascular hemodynamics. Flow assays commonly employ collagen-functionalized substrates, but a lack of standardized methods of surface ligation limits their widespread use as a clinical diagnostic. Here, we report the use of collagen thin films (CTF) in flow assays. Thin films were grown on hydrophobic substrates from type I collagen solutions of increasing concentration (10, 100, and 1000 μg/mL). We found that the corresponding increase in fiber surface area determined the amount of VWF binding and platelet adhesion. The association rate constant (k(a)) of plasma VWF binding at a wall shear stress of 45 dyn/cm(2) was 0.3 × 10(5), 1.8 × 10(5), and 1.6 × 10(5) M(-1) s(-1) for CTF grown from 10, 100, and 1000 μg/mL solutions, respectively. We observed a 5-fold increase in VWF binding capacity with each 10-fold increase in collagen solution concentration. The association rates of Ser1731Thr and His1786Asp VWF mutants with collagen binding deficiencies were 9% and 22%, respectively, of wild-type rates. Using microfluidic devices for blood flow assays, we observed that CTF supported platelet adhesion at a wall shear rate of 1000 s(-1). CTF grown from 10 and 100 μg/mL solutions had variable levels of platelet surface coverage between multiple normal donors. However, CTF substrates grown from 1000 μg/mL solutions had reproducible surface coverage levels (74 ± 17%) between normal donors, and there was significantly diminished surface coverage from two type 1 von Willebrand disease patients (8.0% and 24%). These results demonstrate that collagen thin films are homogeneous and reproducible substrates that can measure dysfunctions in VWF binding and platelet adhesion under flow in a clinical microfluidic assay format.     

Laser-induced thermal bubbles for microfluidic applications

We present a unique bubble generation technique in microfluidic chips using continuous-wave laser-induced heat and demonstrate its application by creating micro-valves and micro-pumps. In this work, efficient generation of thermal bubbles of controllable sizes has been achieved using different geometries of chromium pads immersed in various types of fluid. Effective blocking of microfluidic channels (cross-section 500 × 40 μm(2)) and direct pumping of fluid at a flow rate of 7.2-28.8 μl h(-1) with selectable direction have also been demonstrated. A particular advantage of this technique is that it allows the generation of bubbles at almost any location in the microchannel and thus enables microfluidic control at any point of interest. It can be readily integrated into lab-on-a-chip systems to improve functionality.     

Model-controlled hydrodynamic focusing to generate multiple overlapping gradients of surface-immobilized proteins in microfluidic devices

Historically, it has been difficult to generate accurate and reproducible protein gradients for studies of interactions between cells and extracellular matrix. Here we demonstrate a method for rapid patterning of protein gradients using computer-driven hydrodynamic focusing in a simple microfluidic device. In contrast to published work, we are moving the complexity of gradient creation from the microfluidic hardware to dynamic computer control. Using our method, switching from one gradient profile to another requires only a few hours to devise a new control file, not days or weeks to design and build a new microfluidic device. Fitting existing protein deposition models to our data, we can extract key parameters needed for controlling protein deposition. Several protein deposition models were evaluated under microfluidic flow conditions. A mathematical model for our deposition method allows us to determine the parameters for a protein adsorption model and then predict the final shape of the surface density gradient. Simple and non-monotonic single and multi-protein gradient profiles were designed and deposited using the same device.     

Fabrication of extracellular matrix-coated conductive polypyrrole-poly(l-lactide) fiber-films and their synergistic effect with (nerve growth factor)/(epidermal growth factor) on neurites growth

Non-nerve cell-derived extracellular matrix (ECM) was coated on the aligned porous polypyrrole-poly(l-lactide) (PPy-PLLA) fiber-films with the conductivity of ∼12 mS/m via L929 cells culture and lysing, resulting in ∼10% increase of PC12 cells attachment and ∼26 μm increase of neurites length. The neurite length of ∼149 μm in EGF/NGF group (optimal concentration radio of 12.5/50 (ng/mL)) on aligned and ECM-conjugated fiber-films was significantly larger than ∼94 μm in only NGF group (50 ng/mL), confirming the synergy of EGF, NGF and aligned ECM-conjuaged PPy-PLLA fibers. When differentiated PC12 cells were exerted electrical stimulation (ES) of 100 mV/cm for 4 h/day in 2 day through ECM-PPy-PLLA fiber-films, their neurite length reached to ∼251 μm, significantly larger than ∼149 μm of group without ES, due to the higer expression of related neural proteins in ES group. A simple mechanism was proposed to analyze synergistical effect of ECM, EGF, NGF on axons adhesion and elongation along the aligned ECM-coated fibers under ES condition.     

Surfactant-enhanced liquid-liquid extraction in microfluidic channels with inline electric-field enhanced coalescence

Continuous microfluidic liquid-liquid extraction is realized in a microfluidic device by generating emulsions with large interfacial areas for mass transfer, and subsequently breaking these emulsions using electric fields into easily separated segments of immiscible liquids (plugs). The microfluidic device employs insulated electrodes in a potassium hydroxide-etched channel to create large electric fields (100 kV m(-1)) that drive coalescence of the emulsion phase. The result is a transition from disperse to slug flow that can then readily be separated by gravity. Extractions of phenol and p-nitrophenol from an aqueous to hexane-surfactant solution serve as model systems. In addition to the increased surface area in the emulsion, extraction efficiency is enhanced by reverse micelles resulting from the presence of surfactants. The surfactant concentration is varied approximately 1-10 wt% and a general two-parameter model is developed to quantify the extraction behavior and demonstrate the effectiveness of reverse micelle enhanced extraction.     

Cryptococcus gattii: in vitro susceptibility to photodynamic inactivation

Cryptococus gattii is an emergent primary human pathogen that causes meningismus, papilledema, high intracranial pressure and focal involvement of the central nervous system in immunocompetent hosts. Prolonged antifungal therapy is the conventional treatment, but it is highly toxic, selects for resistant strains, contributes to therapy failure and has a poor prognosis. Photodynamic inactivation (PDI) offers a promising possibility for the alternative treatment of cryptococcosis. The aim of this study was to test the effectiveness of toluidine blue O (TBO) and light-emitting diode (LED) against C. gattii strains with distinct susceptibility profile to antifungal drugs (amphotericin B: 0.015-1.0 μg mL(-1); itraconazole: 0.015-2 μg mL(-1); fluconazole: 4-64 μg mL(-1)). Using 25 μM (6.76 μg mL(-1)) TBO and LED energy density of 54 J cm(-2) these fungal isolates presented variable susceptibility to PDI. The production of reactive oxygen species (ROS)/peroxynitrite was determined, and the catalase and peroxidase activities were measured. After PDI, high amounts of ROS/peroxynitrite are produced and higher catalase and peroxidase activities could be correlated with a lower susceptibility of C. gattii isolates to PDI. These results indicate that PDI could be an alternative to C. gattii growth inhibition, even of isolates less susceptible to classical antifungal drugs, also pointing to mechanisms related to their variable susceptibility behavior.     

Fermentation of Korean Red Ginseng by Lactobacillus plantarum M-2 and Its Immunological Activities

We investigated ginsenoside transformation by fermentation of red ginseng with Lactobacillus plantarum M-2. We also examined the anti-metastasis and immune-stimulating activities of EtOH extracts of fermented red ginseng (FRG-E) in animal and human subjects. Total sugar decreased from 85.5 mg mL(-1) to 44.1 mg mL(-1) with increasing culture time during the fermentation with L. plantarum M-2. Uronic acid content reached a maximum level (534.3 μg mL(-1)) at 3 days of fermentation and decreased thereafter. Ginsenoside metabolites increased from 4,637.0 to 7,581.1 μg mL(-1) after 4 days. The prophylactic intraperitoneal injection of FRG-E (500 μg mouse(-1)) inhibited lung metastasis about 81.1%, while the inhibitory effect against tumor metastasis by treatment of EtOH extract from non-fermented red ginseng (NFRG-E) was 66.9%. Immunoglobulin A (IgA) and G (IgG) levels in the serum of healthy subjects were higher after FRG-E administration than at baseline, whereas NFRG-E induced reductions of these variables related to immunity. At 1 week, the change in IgA level by FRG-E (5.14 mg mL(-1)) was significantly higher than that by NFRG-E (-14.50 mg mL(-1); p < 0.05). It was concluded that the immunological activities of FRG-E were higher than those of NFRG-E, indicating that fermentation helped enhance the immunological activities of red ginseng.