Nanofluidic analytical system integrated with nanochannel open/close valves for enzyme-linked immunosorbent assay.

There have been significant advances in the field of nanofluidics, and novel technologies such as single-cell analysis have been demonstrated. Despite the evident advantages of nanofluidics, fluid control in nanochannels for complicated analyses is extremely difficult because the fluids are currently manipulated by maintaining the balance of driving pressure. To address this issue, the use of valves will be essential. Our group previously developed a nanochannel open/close valve utilizing glass deformation, but this has not yet been integrated into nanofluidic devices for analytical applications. In the present study, a nanofluidic analytical system integrated with multiple nanochannel open/close valves was developed. This system consists of eight pneumatic pumps, seven nanochannel open/close valves combined with piezoelectric actuators, and an ultra-high sensitivity detector for non-fluorescent molecules. For simultaneous actuation of multiple valves, a device holder was designed that prevented deformation of the entire device caused by operating the valves. A system was subsequently devised to align each valve and actuator with a precision of better than 20 µm to permit the operation of valves. The developed analytical system was verified by analyzing IL-6 molecules using an enzyme-linked immunosorbent assay. Fluid operations such as sample injection, pL-level aliquot sampling and flow switching were accomplished in this device simply by opening/closing specific valves, and a sample consisting of approximately 1500 IL-6 molecules was successfully detected. This study is expected to significantly improve the usability of nanofluidic analytical devices and lead to the realization of sophisticated analytical techniques such as single-cell proteomics.

Interleukin-31 promotes fibrosis and T helper 2 polarization in systemic sclerosis.

Systemic sclerosis (SSc) is a chronic multisystem disorder characterized by fibrosis and autoimmunity. Interleukin (IL)-31 has been implicated in fibrosis and T helper (Th) 2 immune responses, both of which are characteristics of SSc. The exact role of IL-31 in SSc pathogenesis is unclear. Here we show the overexpression of IL-31 and IL-31 receptor A (IL-31RA) in dermal fibroblasts (DFs) from SSc patients. We elucidate the dual role of IL-31 in SSc, where IL-31 directly promotes collagen production in DFs and indirectly enhances Th2 immune responses by increasing pro-Th2 cytokine expression in DFs. Furthermore, blockade of IL-31 with anti-IL-31RA antibody significantly ameliorates fibrosis and Th2 polarization in a mouse model of SSc. Therefore, in addition to defining IL-31 as a mediator of fibrosis and Th2 immune responses in SSc, our study provides a rationale for targeting the IL-31/IL-31RA axis in the treatment of SSc.

B Cell Depletion Inhibits Fibrosis via Suppression of Profibrotic Macrophage Differentiation in a Mouse Model of Systemic Sclerosis.

OBJECTIVE: We undertook this study to investigate the effect of B cell depletion on fibrosis in systemic sclerosis (SSc) and its mechanism of action. METHODS: Mice with bleomycin-induced SSc (BLM-SSc) were treated with anti-CD20 antibody, and skin and lung fibrosis were histopathologically evaluated. T cells and macrophages were cocultured with B cells, and the effect of B cells on their differentiation was assessed by flow cytometry. We also cocultured B cells and monocytes from SSc patients and analyzed the correlation between fibrosis and profibrotic macrophage induction by B cells. RESULTS: B cell depletion inhibited fibrosis in mice with BLM-SSc. B cells from mice with BLM-SSc increased proinflammatory cytokine-producing T cells in coculture. In mice with BLM-SSc, B cell depletion before BLM treatment (pre-depletion) inhibited fibrosis more strongly than B cell depletion after BLM treatment (post-depletion) (P < 0.01). However, the frequencies of proinflammatory T cells were lower in the post-depletion group than in the pre-depletion group. This discrepancy suggests that the effect of B cell depletion on fibrosis cannot be explained by its effect on T cell differentiation. On the other hand, profibrotic macrophages were markedly decreased in the pre-depletion group compared to the post-depletion group (P < 0.05). Furthermore, B cells from mice with BLM-SSc increased profibrotic macrophage differentiation in coculture (P < 0.05). In SSc patients, the extent of profibrotic macrophage induction by B cells correlated with the severity of fibrosis (P < 0.0005). CONCLUSION: These findings suggest that B cell depletion inhibits tissue fibrosis via suppression of profibrotic macrophage differentiation in mice with BLM-SSc, providing a new rationale for B cell depletion therapy in SSc.

Cytokine analysis on a countable number of molecules from living single cells on nanofluidic devices.

Analysis of proteins released from living single cells is strongly required in the fields of biology and medicine to elucidate the mechanism of gene expression, cell-cell communication and cytopathology. However, as living single-cell analysis involves fL sample volumes with ultra-small amounts of analyte, comprehensive integration of entire chemical processing for single cells and proteins into spaces smaller than single cells (pL) would be indispensable to prevent dispersion-associated analyte loss. In this study, we proposed and developed a living single-cell protein analysis device based on micro/nanofluidics and demonstrated analysis of cytokines released from living single B cells by enzyme-linked immunosorbent assay. Based on our integration method and technologies including top-down nanofabrication, surface modifications and pressure-driven flow control, we designed and prepared the device where pL-microfluidic- and fL-nanofluidic channels are hierarchically allocated for cellular and molecular processing, respectively, and succeeded in micro/nanofluidic control for manipulating single cells and molecules. 13-unit operations for pL-cellular processing including single-cell trapping and stimulation and fL-molecular processing including fL-volumetry, antigen-antibody reactions and detection were entirely integrated into a microchip. The results suggest analytical performances for countable interleukin (IL)-6 molecules at the limit of detection of 5.27 molecules and that stimulated single B cells secrete 3.41 IL-6 molecules per min. The device is a novel tool for single-cell targeted proteomics, and the methodology of device integration is applicable to other single-cell analyses such as single-cell shotgun proteomics. This study thus provides a general approach and technical breakthroughs that will facilitate further advances in micro/nanofluidics, single-cell life science research, and other fields.

Femtoliter Gradient Elution System for Liquid Chromatography Utilizing Extended Nanofluidics.

A gradient system was developed for the separation of proteins on a femtoliter scale utilizing nanofluidic channels. In the history of chromatography, miniaturization of the separation column has been important for efficient separation and downsizing of instruments. Previously, our group developed a small and highly efficient chromatography system utilizing nanofluidic channels, although a flexible design of the gradient was difficult and separation of proteins was not achieved. Here, we propose a flexible gradient system using standard HPLC pumps and an auxiliary mixer with a simple sample injection system. In contrast to our previous sample injection system using pressure balance, the system enables a femtoliter-scale sample injection which is compatible with gradient elution using HPLC pumps. The system was carefully designed, verified for sample injection and gradient elution, and finally applied to the separation of proteins from model and real samples. This femtoliter-scale, efficient separation system will contribute to omics studies at the single-cell level.

Transport of a Micro Liquid Plug in a Gas-Phase Flow in a Microchannel.

Micro liquid droplets and plugs in the gas-phase in microchannels have been utilized in microfluidics for chemical analysis and synthesis. While higher velocities of droplets and plugs are expected to enable chemical processing at higher efficiency and higher throughput, we recently reported that there is a limit of the liquid plug velocity owing to splitting caused by unstable wetting to the channel wall. This study expands our experimental work to examine the dynamics of a micro liquid plug in the gas phase in a microchannel. The motion of a single liquid plug, 0.4⁻58 nL in volume, with precise size control in 39- to 116-m-diameter hydrophobic microchannels was investigated. The maximum velocity of the liquid plug was 1.5 m/s, and increased to 5 m/s with splitting. The plug velocity was 20% of that calculated using the Hagen-Poiseuille equation. It was found that the liquid plug starts splitting when the inertial force exerted by the fluid overcomes the surface tension, i.e., the Weber number (ratio of the inertial force to the surface tension) is higher than 1. The results can be applied in the design of microfluidic devices for various applications that utilize liquid droplets and plugs in the gas phase.

Rapid alteration of serum interleukin-6 levels may predict the reactivity of i.v. cyclophosphamide pulse therapy in systemic sclerosis-associated interstitial lung disease.

Systemic sclerosis (SSc) is an autoimmune disorder characterized by excessive extracellular matrix deposition. Although SSc-associated interstitial lung disease (ILD) is one of the most important complications as a cause of death in SSc, prediction factors of treatment reactivity in SSc-ILD are still unclear. To assess relationships between interleukin (IL)-6 and reactivity to treatment, we measured serum IL-6 levels in 23 of active SSc-ILD patients under i.v. cyclophosphamide (IVCY) therapy and 20 of stabilized SSc-ILD, using the high-sensitivity enzyme-linked immunoassay system. Serum IL-6 levels in active SSc-ILD patients were significantly higher than those in stabilized SSc-ILD patients. Among active SSc-ILD patients, baseline serum IL-6 levels were not significantly different between IVCY responders and non-responders. Meanwhile, serum IL-6 levels after three IVCY doses out of a total of six were decreased in responders but not in non-responders. Regarding changes of parameters by the three doses of a total of six of IVCY, change in serum IL-6 levels correlated inversely with that in values of pulmonary function test. Thus, the rapid decrease in serum IL-6 levels during a couple of doses may predict the efficacy of IVCY therapy against SSc-ILD.

Integrated Microfluidic Platform with Multiple Functions To Probe Tumor-Endothelial Cell Interaction.

Interaction between tumor and endothelial cells could affect tumor growth and progression and induce drug resistance during cancer therapy. Investigation of tumor-endothelial cell interaction involves cell coculture, protein detection, and analysis of drug metabolites, which are complicated and time-consuming. In this work, we present an integrated microfluidic device with three individual components (cell coculture component, protein detection component, and pretreatment component for drug metabolites) to probe the interaction between tumor and endothelial cells. Cocultured cervical carcinoma cells (CaSki cells) and human umbilical vein endothelial cells (HUVECs) show higher resistance to chemotherapeutic agents than single-cultured cells, indicated by higher cell viability, increased expression of angiogenic proteins, and elevated level of paclitaxel metabolites under coculture conditions. This integrated microfluidic platform with multiple functions facilitates understanding of the interaction between tumor and endothelial cells, and it may become a promising tool for drug screening within an engineered tumor microenvironment.

Contribution of Soluble Forms of Programmed Death 1 and Programmed Death Ligand 2 to Disease Severity and Progression in Systemic Sclerosis.

OBJECTIVE: To determine the function and serum levels of soluble forms of programmed death 1 (sPD-1) and one of its ligands, soluble PD ligand 2 (sPD-L2), in patients with systemic sclerosis (SSc) and in a mouse model of topoisomerase I (topo I)-induced SSc. METHODS: Serum levels of sPD-1 and sPD-L2 in 91 patients with SSc were examined by enzyme-linked immunosorbent assay (ELISA). Expression of PD-1 and PD-L2 on T cells, B cells, and macrophages was quantified by flow cytometry. The effects of blockade of PD-1 and PD-L2 were analyzed by microfluidic ELISA (micro-ELISA), a technique that can measure very low amounts of cytokines. In addition, the effects of sPD-1 and sPD-L2 on disease progression were assessed in mice with topo I-induced SSc. RESULTS: Serum levels of sPD-1 and sPD-L2 were elevated in patients with SSc and correlated with the extent of fibrosis and immunologic abnormalities. Expression levels of PD-1 and PD-L2 were significantly elevated on SSc T cells, B cells, and macrophages. Micro-ELISA analysis of serum samples from patients with SSc showed that PD-L2high B cells had higher levels of interleukin-10 (IL-10) production compared with PD-L2low B cells, indicating that PD-L2 acts as a regulator of T cell cytokine production via cognate interactions with T cells and B cells. In mice with topo I-induced SSc, production of IL-10 by topo I-specific B cells in cultures with T cells and topo I protein was significantly higher than that by conventional B cells, and intraperitoneal injection of recombinant chimeric PD-1-Fc and PD-L2-Fc canceled these enhanced effects. CONCLUSION: These results suggest that sPD-1 and sPD-L2 contribute to disease development in SSc via the regulation of cognate interactions with T cells and B cells.

High-Pressure Acceleration of Nanoliter Droplets in the Gas Phase in a Microchannel.

Microfluidics has been used to perform various chemical operations for pL⁻nL volumes of samples, such as mixing, reaction and separation, by exploiting diffusion, viscous forces, and surface tension, which are dominant in spaces with dimensions on the micrometer scale. To further develop this field, we previously developed a novel microfluidic device, termed a microdroplet collider, which exploits spatially and temporally localized kinetic energy. This device accelerates a microdroplet in the gas phase along a microchannel until it collides with a target. We demonstrated 6000-fold faster mixing compared to mixing by diffusion; however, the droplet acceleration was not optimized, because the experiments were conducted for only one droplet size and at pressures in the 10⁻100 kPa range. In this study, we investigated the acceleration of a microdroplet using a high-pressure (MPa) control system, in order to achieve higher acceleration and kinetic energy. The motion of the nL droplet was observed using a high-speed complementary metal oxide semiconductor (CMOS) camera. A maximum droplet velocity of ~5 m/s was achieved at a pressure of 1⁻2 MPa. Despite the higher fluid resistance, longer droplets yielded higher acceleration and kinetic energy, because droplet splitting was a determining factor in the acceleration and using a longer droplet helped prevent it. The results provide design guidelines for achieving higher kinetic energies in the microdroplet collider for various microfluidic applications.

Selective cell capture and analysis using shallow antibody-coated microchannels.

Demand for analysis of rare cells such as circulating tumor cells in blood at the single molecule level has recently grown. For this purpose, several cell separation methods based on antibody-coated micropillars have been developed (e.g., Nagrath et al., Nature 450, 1235-1239 (2007)). However, it is difficult to ensure capture of targeted cells by these methods because capture depends on the probability of cell-micropillar collisions. We developed a new structure that actively exploits cellular flexibility for more efficient capture of a small number of cells in a target area. The depth of the sandwiching channel was slightly smaller than the diameter of the cells to ensure contact with the channel wall. For cell selection, we used anti-epithelial cell adhesion molecule antibodies, which specifically bind epithelial cells. First, we demonstrated cell capture with human promyelocytic leukemia (HL-60) cells, which are relatively homogeneous in size; in situ single molecule analysis was verified by our rolling circle amplification (RCA) method. Then, we used breast cancer cells (SK-BR-3) in blood, and demonstrated selective capture and cancer marker (HER2) detection by RCA. Cell capture by antibody-coated microchannels was greater than with negative control cells (RPMI-1788 lymphocytes) and non-coated microchannels. This system can be used to analyze small numbers of target cells in large quantities of mixed samples.

Development of a microfluidic platform for single-cell secretion analysis using a direct photoactive cell-attaching method.

A precise understanding of individual cellular processes is essential to meet the expectations of most advanced cell biology. Therefore single-cell analysis is considered to be one of possible approach to overcome any misleading of cell characteristics by averaging large groups of cells in bulk conditions. In the present work, we modified a newly designed microchip for single-cell analysis and regulated the cell-adhesive area inside a cell-chamber of the microfluidic system. By using surface-modification techniques involving a silanization compound, a photo-labile linker and the 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer were covalently bonded on the surface of a microchannel. The MPC polymer was utilized as a non-biofouling compound for inhibiting non-specific binding of the biological samples inside the microchannel, and was selectively removed by a photochemical reaction that controlled the cell attachment. To achieve the desired single-macrophage patterning and culture in the cell-chamber of the microchannel, the cell density and flow rate of the culture medium were optimized. We found that a cell density of 2.0 × 10(6) cells/ml was the appropriate condition to introduce a single cell in each cell chamber. Furthermore, the macrophage was cultured in a small size of the cell chamber in a safe way for 5 h at a flow rate of 0.2 µl/min under the medium condition. This strategy can be a powerful tool for broadening new possibilities in studies of individual cellular processes in a dynamic microfluidic device.

Experimental investigation of droplet acceleration and collision in the gas phase in a microchannel.

We developed a novel microfluidic system, termed a micro-droplet collider, by utilizing the spatial-temporal localized liquid energy to realize chemical processes, which achieved rapid mixing between droplets having a large volume ratio by collision. In this paper, in order to clarify the characteristics of the micro-droplet collider, dynamics of droplet acceleration, stationary motion and collision in the gas phase in a microchannel were experimentally investigated with visualized images using a microscope equipped with a high-speed camera. The maximum velocity of 450 mm s(-1) and acceleration of 1500 m s(-2) of a 1.6 nL water droplet were achieved at an air pressure of 100 kPa. Measurement results of dynamic contact angles of droplets indicated that wettability of the surface played an important role in the stability of droplet acceleration and collision. We found that the bullet droplet penetrated into the target droplet at collision, which differed from bulk scale. The deformation of the droplet was strongly suppressed by the channel structure, thus stable collision and efficient utilization of the droplet energy were possible. These results are useful for estimating the localized energy, for improving the system in order to realize extreme performance, and for extending the applications of microfluidic devices.

In situ assembly, regeneration and plasmonic immunosensing of a Au nanorod monolayer in a closed-surface flow channel.

Herein, a simple and effective approach is reported for the in situ generation and regeneration of a Au nanorod (AuNR) monolayer inside a glass/silica-based, closed-surface flow channel. The density of the AuNR monolayer in the flow channel can be easily modified by varying the concentration of the AuNR and the cetyltrimethylammonium bromide as well as the incubation time. The fabricated AuNR monolayer in the flow channels was stable under harsh conditions, such as in extreme pH, organic solvents and at a fast flow rate. In addition, the flow channel could be reused by removing the immobilized AuNRs via the injection of diluted aqua regia or potassium iodide; the AuNR monolayer can subsequently be regenerated. The AuNRs in the closed flow channel were further exploited as a label-free detection method for a clinical biomarker, neutrophil gelatinase-associated lipocalin (NGAL), based on single-nanoparticle plasmonic assay. The corresponding limit of detection for NGAL was measured to be 8.5 ng mL(-1) (~340 pM) based on a signal-to-noise ratio of 3. The estimated recovery of NGAL in human serum and urine was higher than 80%, which indicates that this technique could potentially be used for the diagnosis of acute kidney injury.

Sensitive gas analysis system on a microchip and application for on-site monitoring of NH3 in a clean room.

A portable, highly sensitive, and continuous ammonia gas monitoring system was developed with a microfluidic chip. The system consists of a main unit, a gas pumping unit, and a computer which serves as an operation console. The size of the system is 45 cm width × 30 cm depth × 30 cm height, and the portable system was realized. A highly efficient and stable extraction method was developed by utilizing an annular gas/liquid laminar flow. In addition, a stable gas/liquid separation method with a PTFE membrane was developed by arranging a fluidic network in three dimensions to achieve almost zero dead volume at the gas/liquid extraction part. The extraction rate was almost 100% with a liquid flow rate of 3.5 µL/min and a gas flow rate of 100 mL/min (contact time of ~15 ms), and the concentration factor was 200 times by calculating the NH(3) concentration (w/w unit) in the gas and liquid phases. Stable phase separation and detection was sustained for more than 3 weeks in an automated operation, which was sufficient for the monitoring application. The lower limit of detection calculated based on a signal-to-noise ratio of 3 was 84 ppt, which showed good detectability for NH(3) analysis. We believe that our system is a very powerful tool for gas analysis due to the advantages of portable size, high sensitivity, and continuous monitoring, and it is particularly useful in the semiconductor field.

An efficient surface modification using 2-methacryloyloxyethyl phosphorylcholine to control cell attachment via photochemical reaction in a microchannel.

This report describes a direct approach for cell micropatterning in a closed glass microchannel. To control the cell adhesiveness inside the microchannel, the application of an external stimulus such as ultraviolet (UV) was indispensible. This technique focused on the use of a modified 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, which is known to be a non-biofouling compound that is a photocleavable linker (PL), to localize cells via connection to an amino-terminated silanized surface. Using UV light illumination, the MPC polymer was selectively eliminated by photochemical reaction that controlled the cell attachment inside the microchannel. For suitable cell micropatterning in a microchannel, the optimal UV illumination time and concentration for cell suspension were investigated. After selective removal of the MPC polymer through the photomask, MC-3T3 E1 cells and vascular endothelial cells (ECs) were localized only to the UV-exposed area. In addition, the stability of patterned ECs was also confirmed by culturing for 2 weeks in a microchannel under flow conditions. Furthermore, we employed two different types of cells inside the same microchannel through multiple removal of the MPC polymer. ECs and Piccells were localized in both the upper and down streams of the microchannel, respectively. When the ECs were stimulated by adenosine triphosphate (ATP), NO was secreted from the ECs and could be detected by fluorescence resonance energy transfer (FRET) in Piccells, which is a cell-based NO indicator. This technique can be a powerful tool for analyzing cell interaction research.

Cell culture and motility study on a polymer surface with a nanometer-scaled stripe structure.

We developed a large cell culture surface with a nanostripe structure by paving polydimethylsiloxane (PDMS) replicas of a glass mold. The stripe structure has a height of 180 nm and top width of 500 nm with 400-nm intervals between stripes. Human stomach cancer SH-10-TC cells cultured on the surface changed their morphology to elongated shapes parallel to the nanostripes. In addition, cell motility parallel to the stripes was greatly enhanced. These findings strongly suggest that the nanostripe structure affected the cell physiology.

Culture and leukocyte adhesion assay of human arterial endothelial cells in a glass microchip.

Cells are frequently exploited as processing components for integrated chemical systems, such as biochemical reactors and bioassay systems. By culturing vascular endothelial cells (ECs) in integrated chemical devices, vascular models have also been fabricated. Here, we utilized a thermally fused-glass microchip which is chemically and physically stable and favorable for optical detections, and cultured human arterial ECs (HAECs) in it. HAECs reached confluence within 4 days. Survival and tolerance for high shear stress (25 dyn/cm2) of the HAECs were confirmed. Furthermore, HAECs responded to inflammatory cytokine, tumor necrosis facor-alpha (TNF-alpha) and attached to more leukocyte cell line, HL-60 cells than unstimulated HAECs. Our developed device can be applied as a human arterial model, and we propose it as a new method for vascular studies.

Application of a micro multiphase laminar flow on a microchip for extraction and determination of derivatized carbamate pesticides.

Determination of carbamate pesticides such as carbaryl, carbofuran, propoxur and bendiocarb was demonstrated on a microchip with newly designed microchannels developed for efficient solvent extraction. The pesticides were hydrolyzed to corresponding naphthols, coupled with p-nitrobenzenediazonium fluoroborate reagent, and then extracted into 1-butanol as colored azo derivatives and detected with a thermal lens microscope. Optimum flow rates for the aqueous and organic phases were evaluated in the continuous-flow chemical process established in the microchip. The calibration lines showed good linearity in the range of concentrations of 0.03 - 3 ppm (10(-7) - 10(-5) M) and a mass detection limit down to a nanogram level was achieved that is at least two orders of magnitude lower than the LODs for conventional spectrophotometric methods. Azo derivatives of the pesticides were successfully separated and identified by micellar electrokinetic chromatography (MEKC) using a sample prepared on a bulk scale.

Microchip-based liquid-liquid extraction for gas-chromatography analysis of amphetamine-type stimulants in urine.

A microchip-based liquid-liquid extraction for the gas chromatography analysis of urine for amphetamine-type stimulants has been developed. Partially modified microchannels with the capillarity restricted modification (CARM) method were employed for stabilizing the interface consisting of 1-chlorobutane and alkalized urine. Reliability of the microchip-based extraction was evaluated with respect to linearity, trueness and precision. As a practical demonstration, methoxyphenamine hydrochloride (50 mg) was administered to three healthy volunteers, and the concentration of methoxyphenamine in their urine was determined by both methods for comparison. This study showed the potential of pressure-driven microfluidics to contribute to the rapid automation analysis in forensic toxicology.