Determination of standard molar volume of 1-hexyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide on titanium dioxide surface.

The fluids near the solid substrate display different properties compared to the bulk fluids owing to the asymmetric interaction between the fluid and substrate; however, to the best of our knowledge, no work has been conducted to determine the interfacial properties of fluids experimentally. In this work, we combined a pycnometer with experimental measurements and data processing to determine the standard thermodynamic properties of interfacial fluids for the first time. In the study, 1-hexyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([Hmim][NTf2]) and titanium dioxide (P25) were chosen as the probes to prove the concept. It was found that, with the combination of the Gay-Lussac pycnometer and the colligative law, together with selecting a suitable solvent, it is possible and reliable to determine the standard molar volume of the immobilized [Hmim][NTf2]. Compared to the bulk phase, the molar volumes of [Hmim][NTf2] on the P25 surface reduce by 20.8%-23.7% at temperatures from 293.15 to 323.15 K, and the reduction degrees decrease with increasing temperatures. The newly determined standard thermodynamic data was used to obtain the model parameters of hybrid electrolyte perturbed-chain statistical associating fluid theory density functional theory (ePC-SAFT-DFT), and further predictions of the density of interfacial ionic liquids with different film thicknesses were proved to be reliable in comparison with the experiment results.

Enhanced CO2 Capture through SAPO-34 Impregnated with Ionic Liquid.

The concurrent utilization of an adsorbent and absorbent for carbon dioxide (CO2) adsorption with synergistic effects presents a promising technique for CO2 capture. Here, 1-butyl-3-methylimidazole acetate ([Bmim][Ac]), with a high affinity for CO2, and the molecular sieve SAPO-34 were selected. The impregnation method was used to composite the hybrid samples of [Bmim][Ac]/SAPO-34, and the pore structure and surface property of prepared samples were characterized. The quantity and kinetics of the sorbed CO2 for loaded samples were measured using thermogravimetric analysis. The study revealed that SAPO-34 could retain its pristine structure after [Bmim][Ac] loading. The CO2 uptake of the loaded sample was 1.879 mmol g-1 at 303 K and 1 bar, exhibiting a 20.6% rise compared to that of the pristine SAPO-34 recording 1.558 mmol g-1. The CO2 uptake kinetics of the loaded samples were also accelerated, and the apparent mass transfer resistance for CO2 sorption was significantly reduced by 11.2% compared with that of the pure [Bmim][Ac]. The differential scanning calorimetry method revealed that the loaded sample had a lower CO2 desorption heat than that of the pure [Bmim][Ac], and the CO2 desorption heat of the loaded samples was between 30.6 and 40.8 kJ mol-1. The samples exhibited good cyclic stability. This material displays great potential for CO2 capture applications, facilitating the reduction of greenhouse gas emissions.

ß-Asarone Protects PC12 Cells Against Hypoxia-Induced Injury Via Negatively Regulating RPPH1/MiR-542-3p/DEDD2 Axis.

Hypoxic injury to the brain is very intricate under the control of biochemical reactions induced by various factors and mechanisms. Long non-coding RNAs (lncRNAs) have already been revealed to affect pathological processes in the nervous system of different degrees. This research aimed to investigate the mechanisms implicated in hypoxic brain injury. ß-Asarone mitigated the decrease of cell viability, superoxide dismutase activity, and mitochondrial membrane potential, as well as the increase of cell apoptosis, lactate dehydrogenase release, malondialdehyde content, and reactive oxidative species production by cobalt chloride. LncRNA ribonuclease P RNA component H1 (RPPH1) was discovered to be highly expressed in hypoxia-induced PC12 cells, and ß-Asarone addition led to a decline in RPPH1 expression. RPPH1 overexpression reversed the effect of ß-Asarone on hypoxia-induced injury in PC12 cells. Furthermore, we proved that RPPH1 could sponge miR-542-3p. Subsequently, death effector domain containing 2 (DEDD2) was proven as the downstream gene of RPPH1/miR-542-3p axis. Eventually, the whole regulation mechanism of RPPH1/miR-542-3p/DEDD2 axis was testified through rescue assays. The impacts of ß-Asarone on hypoxia-induced PC12 cells could be countervailed by RPPH1 augment, which was also discovered to be neutralized in response to miR-542-3p overexpression or DEDD2 depletion. These findings offered a novel perspective for understanding neuroprotection.

Flow induced adherens junction remodeling driven by cytoskeletal forces.

Adherens junctions (AJs) are a key structural component for tissue organization and function. Under fluid shear stress, AJs exhibit dynamic assembly/disassembly, but how shear stress couples to AJs is unclear. In MDCK cells we measured simultaneously the forces in cytoskeletal α-actinin and the density and length of AJs using a genetically coded optical force sensor, actinin-sstFRET, and fluorescently labeled E-cadherin (E-cad). We found that shear stress of 0.74dyn/cm2 for 3h significantly enhanced E-cad expression at cell-cell contacts and this phenomenon has two phases. The initial formation of segregated AJ plaques coincided with a decrease in cytoskeletal tension, but an increase in tension was necessary for expansion of the plaques and the formation of continuous AJs in the later phase. The changes in cytoskeletal tension and reorganization appear to be an upstream process in response to flow since it occurred in both wild type and dominant negative E-cad cells. Disruption of F-actin with a Rho-ROCK inhibitor eliminated AJ growth under flow. These results delineate the shear stress transduction paths in cultured cells, which helps to understand pathology of a range of diseases that involve dysfunction of E-cadherin.

Intracellular forces during guided cell growth on micropatterns using FRET measurement.

Interaction of cells with extracellular matrix (ECM) regulates cell shape, differentiation and polarity. This effect has been widely observed in cells grown on substrates with various patterned features, stiffness and surface chemistry. It has been postulated that mechanical confinement of cells by the substrate causes a redistribution of tension in cytoskeletal proteins resulting in cytoskeletal reorganization through force sensitive pathways. However, the mechanisms for force transduction during reorganization remain unclear. In this study, using FRET based force sensors we have measured tension in an actin cross-linking protein, α-actinin, and followed reorganization of actin cytoskeleton in real time in HEK cells grown on patterned substrates. We show that the patterned substrates cause a redistribution of tension in α-actinin that coincides with cytoskeleton reorganization. Higher tension was observed in portions of cells where they form bridges across inhibited regions of the patterned substrates; the attachment to the substrate is found to release tension. Real time measurements of α-actinin tension and F-actin arrangement show that an increase in tension coincides with formation of F-actin bundles at the cell periphery during cell-spreading across inhibited regions, suggesting that mechanical forces stimulate cytoskeleton enhancement. Rho-ROCK inhibitor (Y27632) causes reduction in actinin tension followed by retraction of bridged regions. Our results demonstrate that changes in cell shape and expansion over patterned surfaces is a force sensitive process that requires actomyosin contractile force involving Rho-ROCK pathway.

Direct observation of α-actinin tension and recruitment at focal adhesions during contact growth.

Adherent cells interact with extracellular matrix via cell-substrate contacts at focal adhesions. The dynamic assembly and disassembly of focal adhesions enables cell attachment, migration and growth. While the influence of mechanical forces on the formation and growth of focal adhesions has been widely observed, the force loading on specific proteins at focal adhesion complex is not clear. By co-expressing force sensitive α-actinin FRET probes and fluorescence labeled paxillin in MDCK cells, we have simultaneously observed the time-dependent changes in tension in α-actinin and the dynamics of focal adhesion during cell migration. We show that increase in tension in α-actinin at the focal adhesion coincides with elongation of the adhesion in its growth phase. The enlargement of focal adhesion is through a force sensitive recruitment of α-actinin and paxillin to the adhesion sites. Changes in α-actinin tension and correlated relocation of α-actinin in an active adhesion also guide the growth direction of the adhesion. The results support the model that cytoskeletal tension is coupled to focal adhesion via the linking protein, α-actinin at the adhesion complex. Lysophosphatidic acid caused an immediate increase in α-actinin tension followed by drastic focal adhesion formation and elongation. Application of Rho-ROCK inhibitor, Y27632, resulted in reversible reduction in tension in α-actinin and disassociation of focal adhesion, suggesting the involvement of myosin-II mediated contractile force in the focal adhesion dynamics. These findings suggest that α-actinin not only serves as a physical linker between cytoskeleton and integrin, but also participates in force transmission at adhesion sites to facilitate adhesion׳s growth.

Interplay between cytoskeletal stresses and cell adaptation under chronic flow.

Using stress sensitive FRET sensors we have measured cytoskeletal stresses in α-actinin and the associated reorganization of the actin cytoskeleton in cells subjected to chronic shear stress. We show that long-term shear stress reduces the average actinin stress and this effect is reversible with removal of flow. The flow-induced changes in cytoskeletal stresses are found to be dynamic, involving a transient decrease in stress (phase-I), a short-term increase (3-6 min) (Phase-II), followed by a longer-term decrease that reaches a minimum in ~20 min (Phase-III), before saturating. These changes are accompanied by reorganization of the actin cytoskeleton from parallel F-actin bundles to peripheral bundles. Blocking mechanosensitive ion channels (MSCs) with Gd(3+) and GsMTx4 (a specific inhibitor) eliminated the changes in cytoskeletal stress and the corresponding actin reorganization, indicating that Ca(2+) permeable MSCs participate in the signaling cascades. This study shows that shear stress induced cell adaptation is mediated via MSCs.

Assay for molecular transport across gap junction channels in one-dimensional cell arrays.

Transport across gap junction channels (GJCs) between neighboring cells is critical to synchronizing cell's electrical and metabolic activities and maintaining cell homeostasis. Here we present a non-invasive microfluidic method to measure molecular diffusion across GJCs in multiple 1D cell arrays in real time. Using the chip, selective loading of a membrane permeant fluorescence dye (carboxyfluorescein) in Normal Rat Kidney (NRK) cells shows that the dye was able to diffuse through three cells along single cell chains in ∼35 minutes. Application of 100 µM 2-aminoethoxydiphenyl borate (2-APB) reversibly inhibits connexin-43 gap junctions in NRK cells; 0.8 mM 1-heptanol inhibits the diffusion partially. The method offers rapid exchange of reagents, enabling sequential screening of multiple gap junction specific drugs with only one preparation of cells. It is capable of measuring gap junction mediated diffusion between single cells.

Microfluidic devices for characterizing the agonist of formyl peptide receptor in RBL-FPR cells.

The human formyl peptide receptor (FPR) plays an important role in inflammation and immunity. Finding of specific agonists and antagonists of FPR may provide potential therapeutic agents for FPR related disorders. The binding of agonist by FPR induces a cascade of G protein-mediated signaling events leading to neutrophil chemotaxis, intracellualr calcium mobilization, FPR ligand uptake and so on. This work proposed a microfluidic-based method to characterize FPR-related cellular events in response to small peptides, N-formyl-Met-Leu-Phe (fMLF), in rat basophilic leukemia cell line RBL-2H3 expressing human FPR (RBL-FPR). The results showed that fMLF triggered chemotaxis, calcium mobilization and FPR ligand uptake in RBL-FPR cells, indicating the potential role of FPR agonist. The chemotaxis index and the calcium mobilization intensity increased but the time course of calcium mobilization decreased, as the rising of fMLF concentration. The basic agreement between the microfluidic results and the previous studies demonstrated good feasibility of the microfluidic method for characterization of FPR agonist. Microfluidic technology displays significant advantages over traditional methods in terms of sample consumption and assay time. It also facilitates experimental process and real-time observation of cellular responses at single cell resolution.

Droplet-based microfluidic system for individual Caenorhabditis elegans assay.

A droplet-based microfluidic system integrating a droplet generator and a droplet trap array is described for encapsulating individual Caenorhabditis elegans into a parallel series of droplets, enabling characterization of the worm behavior in response to neurotoxin at single-animal resolution.

Cell-based high content screening using an integrated microfluidic device.

High content screening (HCS) has quickly established itself as a core technique in the early stage of drug discovery for secondary compound screening. It allows several independent cellular parameters to be measured in a single cell or populations of cells in a single assay. In this work, we describe high content screening for the multiparametric measurement of cellular responses in human liver carcinoma (HepG2) cells using an integrated microfluidic device. This device consists of multiple drug gradient generators and parallel cell culture chambers, in which the processes of liquid dilution and diffusion, micro-scale cell culture, cell stimulation and cell labeling can be integrated into a single device. The simple assay provides multiparametric measurements of plasma membrane permeability, nuclear size, mitochondrial transmembrane potential and intracellular redox states in anti-cancer drug-induced apoptosis of HepG2 cells. The established platform is able to rapidly extract the maximum of information from tumor cells in response to several drugs varying in concentration, with minimal sample and less time, which is very useful for basic biomedical research and cancer treatment.

Parallel microfluidic networks for studying cellular response to chemical modulation.

A microfluidic chip featuring parallel gradient-generating networks etched on glass plate was designed and fabricated. The dam and weir structures were fabricated to facilitate cell positioning and seeding, respectively. The microchip contains five gradient generators and 30 cell chambers where the resulted concentration gradients of drugs are delivered to stimulate the on-chip cultured cells. This microfluidics exploits the advantage of lab-on-a-chip technology by integrating the generation of drug concentration gradients and a series of cell operations including seeding, culture, stimulation and staining into a chip. Steady parallel concentration gradients were generated by flowing two fluids in each network. The microchip described above was applied in studying the role of reduced glutathione (GSH) in MCF-7 cells' chemotherapy sensitivity. The parental breast cancer cell line, MCF-7 and the derived adriamycin resistant cell line MCF-7(adm) were treated with concentration gradients of arsenic trioxide (ATO) and N-acetyl cysteine (NAC) for GSH modulation, followed by exposure to adriamycin. The intracellular GSH level and cell viability were assessed by fluorescence image analysis. GSH levels of both cell lines were down-regulated upon ATO treatment and up-regulated upon NAC treatment. For both cell lines, suppression of intracellular GSH by treatment with ATO has been shown to increase chemotherapy sensitivity; conversely, elevation of intracellular GSH by treatment with NAC leads to increased drug resistance. The results indicated that high intracellular GSH level has negative effect on chemotherapy sensitivity, while depletion of cellular GSH may serve as an effective way to improve chemotherapy sensitivity. The integrated microfluidic chip is able to perform multiparametric pharmacological profiling with easy operation, thus, holds great potential for extrapolation to the high-content drug screening.

Fabrication of two-weir structure-based packed columns for on-chip solid-phase extraction of DNA.

Microchip-based packed column SPE of DNA was performed using the microfabricated two-weir structure within a microchannel. We developed two methods to fabricate the two-weir structured glass chips: a "two-side etching/alignment" method and a simplified "one-side/two-step etching" method. The former method required a straightforward alignment step, while the latter approach comprised a simplified wet etching process using paraffin wax as the temporary protective layer. Both methods were convenient and rapid as compared to the previous approaches. Through a reversibly sealed bead-introduction channel, beads can be fed into or out of the chip columns, thus enabling refreshment of the packing materials. Using the proposed chip columns, highly efficient lambda-DNA extractions (average recovery >80%) were performed with good chip-to-chip reproducibility (RSD <10%). The on-chip SPE procedure was completed within 15 min at the flow rate of 3 microL/min and the bulk of the loaded DNA was eluted within a small volume of approximately 8 microL. Application of the microchip-based packed columns was demonstrated by purifying PCR-amplifiable genomic DNA from human hepatocellular carcinoma (HepG2) cells and human whole blood samples.

Characterizing doxorubicin-induced apoptosis in HepG2 cells using an integrated microfluidic device.

Apoptosis has now established its importance in numerous areas of biology and is recently receiving great attention as an important topic related to the development of diseases. In this work, an integrated microfluidic device was developed to characterize doxorubicin-induced apoptosis in human hepatocellular carcinoma (HepG2) cells. A continuous concentration gradient of stimulator (doxorubicin) was generated in the upstream network and used to perfuse downstream cultured HepG2 cells. The appropriate fluorescent dyes were introduced into cells from the inlets connected to the cell culture chambers, allowing one to distinguish apoptotic cells from nonapoptotic or necrotic cells. The resultant fluorescence of cellular population was monitored and quantified with single-cell resolution to infer the apoptosis process being studied. The feasibility of studying apoptosis was demonstrated by measuring several apoptotic events, including morphological alterations, plasma membrane phosphatidylserine externalization, and mitochondrial membrane potential collapse. This microfluidic device, integrating the cell culture, stimulation, staining, and washing steps into a single device, can simultaneously generate a number of experimental conditions and investigate multiple parameters relating stimulation to apoptosis. It offers a unique platform to characterize various cellular responses in a high-throughput fashion, which is otherwise impossible with conventional methods.

Integration of nanoporous membranes for sample filtration/preconcentration in microchip electrophoresis.

Microfluidic devices integrating membrane-based sample preparation with electrophoretic separation are demonstrated. These multilayer devices consist of 10 nm pore diameter membranes sandwiched between two layers of PDMS substrates with embedded microchannels. Because of the membrane isolation, material exchange between two fluidic layers can be precisely controlled by applied voltages. More importantly, since only small molecules can pass through the nanopores, the integrated membrane can serve as a filter or a concentrator prior to microchip electrophoresis under different design and operation modes. As a filter, they can be used for separation and selective injection of small analytes from sample matrix. This has been effectively applied in rapid determination of reduced glutathione in human plasma and red blood cells without any off-chip deproteinization procedure. Alternatively, in the concentrator mode, they can be used for online purification and preconcentration of macromolecules, which was illustrated by removing primers and preconcentrating the product DNA from a PCR product mixture.

Parallel analysis of biomolecules on a microfabricated capillary array chip.

This paper focused on a self-developed microfluidic array system with microfabricated capillary array electrophoresis (mu-CAE) chip for parallel chip electrophoresis of biomolecules. The microfluidic array layout consists of two common reservoirs coupled to four separation channels connected to sample injection channel on the soda-lime glass substrate. The excitation scheme for distributing a 20 mW laser beam to separation channels in an array is achieved. Under the control of program, the sample injection and separation in multichannel can be achieved through six high-voltage modules' output. A CCD camera was used to monitor electrophoretic separations simultaneously in four channels with LIF detection, and the electropherograms can be plotted directly without reconstruction by additional software. Parallel multichannel electrophoresis of series biomolecules including amino acids, proteins, and nucleic acids was performed on this system and the results showed fine reproducibility.

Analysis of multiplex PCR fragments with PMMA microchip.

Microchip electrophoresis is a promising technique for analysis of bio-molecules. It has the advantages of fast analysis, high sensitivity, high resolution and low-cost of samples. Plastic chip has the potential of mass production for clinical use for its advantages in biocompatibility and low cost. In this work, the method for fabrication of poly(methyl methacrylate) (PMMA) chip was described, and conditions for DNA separation were investigated with the chip. The PMMA microchip was used for detection of multiplex PCR products of 18 and 36 cases with SARS and hepatitis B virus infection under optimized separation conditions. Microchip electrophoresis showed higher sensitivity, higher resolution and less time consumption when compared with gel electrophoresis. The microchip electrophoresis with PMMA chip provided a rapid, sensitive and reliable method for analysis of multiplex PCR products.

Microfluidic devices for the analysis of apoptosis.

Apoptosis is the outcome of a metabolic cascade that results in cell death in a controlled manner. Due to its important role in maintaining balance in organisms, in mechanisms of diseases, and tissue homeostasis, apoptosis is of great interest in the emerging fields of systems biology. Research into cell death regulation and efforts to model apoptosis processes have become powerful drivers for new technologies to acquire ever more comprehensive information from cells and cell populations. The microfluidic technology promises to integrate and miniaturize many bioanalytical processes, which offers an alternative platform for the analysis of apoptosis. This review aims to highlight the recent developments of microfluidic devices in measuring the hallmarks as well as the dynamic process of cellular apoptosis. The potential capability and an outlook of microfluidic devices for the study of apoptosis are addressed.

Simultaneous and ultrarapid determination of reactive oxygen species and reduced glutathione in apoptotic leukemia cells by microchip electrophoresis.

A microchip electrophoresis method coupled with laser-induced fluorescence (LIF) detection was established for simultaneous determination of two kinds of intracellular signaling molecules (reactive oxygen species, ROS, and reduced glutathione, GSH) related to apoptosis and oxidative stress. As the probe dihydrorhodamine-123 (DHR-123) can be converted intracellularly by ROS to the fluorescent rhodamine-123 (Rh-123), and the probe naphthalene-2,3-dicarboxaldehyde (NDA) can react quickly with GSH to produce a fluorescent adduct, rapid determination of Rh-123 and GSH was achieved on a glass microchip within 27 s using a 20 mM borate buffer (pH 9.2). The established method was tested to measure the intracellular ROS and GSH levels in acute promyelocytic leukemia (APL)-derived NB4 cells. An elevation of intracellular ROS and depletion of GSH were observed in apoptotic NB4 cells induced by arsenic trioxide (As(2)O(3)) at low concentration (1-2 microM). Buthionine sulfoximine (BSO), in combination with As(2)O(3) enhanced the decrease of reduced GSH to a great extent. The combined treatment of As(2)O(3) and hydrogen peroxide (H(2)O(2)) led to an inverse relationship between the concentrations of ROS and GSH obtained, showing the proposed method can readily evaluate the generation of ROS, which occurs simultaneously with the consumption of the inherent antioxidant.