Quantitative characterization of liquids flowing in geometrically controlled sub-100 nm nanofluidic channels.

With development of nanotechnologies, applications exploiting nanospaces such as single-molecule analysis and high-efficiency separation have been reported, and understanding properties of fluid flows in 101 nm to 102 nm scale spaces becomes important. Nanofluidics has provided a platform of nanochannels with defined size and geometry, and revealed various unique liquid properties including higher water viscosity with dominant surface effects in 102 nm spaces. However, experimental investigation of fluid flows in 101 nm spaces is still difficult owing to lack of fabrication procedure for 101 nm nanochannels with smooth walls and precisely controlled geometry. In the present study, we established a top-down fabrication process to realize fused-silica nanochannels with 101 nm scale size, 100 nm roughness and rectangular cross-sectional shape with an aspect ratio of 1. Utilizing a method of mass flowmetry developed by our group, accurate measurements of ultra-low flow rates in sub-100 nm nanochannels with sizes of 70 nm and 100 nm were demonstrated. The results suggested that the viscosity of water in these sub-100 nm nanochannels was approximately 5 times higher than that in the bulk, while that of dimethyl sulfoxide was similar to the bulk value. The obtained liquid permeability in the nanochannels can be explained by a hypothesis of loosely structured liquid phase near the wall generated by interactions between the surface silanol groups and protic solvent molecules. The present results suggest the importance of considering the species of solvent, the surface chemical groups, and the size and geometry of nanospaces when designing nanofluidic devices and membranes.

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.

Femtoliter-Droplet Mass Spectrometry Interface Utilizing Nanofluidics for Ultrasmall and High-Sensitivity Analysis.

In the fields of biology and medicine, comprehensive protein analysis at the single-cell level utilizing mass spectrometry (MS) with pL sample volumes and zmol to amol sensitivity is required. Our group has developed nanofluidic analytical pretreatment methods that exploit nanochannels for downsizing chemical unit operations to fL-pL volumes. In the field of analytical instruments, mass spectrometers have advanced to achieve ultrahigh sensitivity. However, a method to interface between fL-pL pretreatments and mass spectrometers without sample loss and dispersion is still challenging. In this study, we developed an MS interface utilizing nanofluidics to achieve high-sensitivity detection. After charging analyte molecules by an applied voltage through an electrode, the liquid sample was converted to fL droplets by a nanofluidic device. Considering the inertial force that acts on the droplets, the droplets were carried with a controlled trajectory, even in turbulent air flow, and injected into a mass spectrometer with 100% efficiency. A module for heat transfer was designed and constructed, by which all of the injected droplets were vaporized to produce gas-phase ions. The detection of caffeine ions was achieved at a limit of detection of 1.52 amol, which was 290 times higher than a conventional MS interface by electrospray ionization with sample dispersion combined with a similar mass spectrometer. Therefore, sensitivity that was 2 orders of magnitude higher could be realized due to the 100% sample injection rate. The present study provides a new methodology for the analysis of ultrasmall samples with high-sensitivity, such as protein molecules produced from a single cell.

Characterization of pressure-driven water flows in nanofluidic channels by mass flowmetry.

With developments in analytical devices promoted by nanofluidics, estimation of the flow rate in a nanochannel has become important to calculate volumes of samples and reagents in chemical processing. However, measurement of the flow rate in nanospaces remains challenging. In the present study, a mass flowmetry system was developed, and the flow rate of water by pressure-driven flow in a fused-silica nanochannel was successfully measured in picoliters per second. We revealed that the water flow rate is dependent on the viscosity significantly increased in a square nanochannel with 102 nm width and depth (3.6 times higher than the bulk viscosity for a representative channel size of 190 nm) and slightly increased in a plate nanochannel with micrometer-scale width and 102 nm depth (1.3 times higher for that of 234 nm), because of dominant surface effects. The developed method and results obtained will greatly contribute to nanofluidics and other related fields.

Accelerated protein digestion and separation with picoliter volume utilizing nanofluidics.

Single cell analyses can provide critical biological insight into cellular heterogeneity. In particular, the proteome, which governs cell functions, is much more difficult to analyze because it is principally impossible to amplify proteins compared to nucleic acids. The most promising approach to single cell proteomics is based on the liquid chromatography mass spectrometry (LC-MS) platform. However, pretreatments before MS detection have two critical issues for single cell analysis: analyte loss as a result of adsorption and artifacts due to the duration of analysis. This is a serious problem because single cells have a limited number of protein molecules and a small volume. To solve these issues, we developed an integrated nanofluidic device to manipulate samples on a femtoliter to picoliter (fL-pL) scale to achieve high-throughput analysis via suppressing analyte loss. This device can perform tryptic digestion, chromatographic separation, and non-labeled detection with high consistency. In addition, we introduced an open/close valve by physical deformation of glass on a nanometer scale to independently modify the nanochannel surfaces and control sample aliquots. The injection system equipped with this valve achieved an injection volume of 1.0 ± 0.1 pL. By using this integrated device, we found that the chromatogram of bulk-digestion for 12 hours resembled that of 15 min-digestion in the nanochannel, which indicated that these conditions reached a similar state of digestion. Therefore, an integrated device for ultra-fast protein analysis was developed on a 1 pL scale for the first time.

Single-cell-level protein analysis revealing the roles of autoantigen-reactive B lymphocytes in autoimmune disease and the murine model.

Despite antigen affinity of B cells varying from cell to cell, functional analyses of antigen-reactive B cells on individual B cells are missing due to technical difficulties. Especially in the field of autoimmune diseases, promising pathogenic B cells have not been adequately studied to date because of its rarity. In this study, functions of autoantigen-reactive B cells in autoimmune disease were analyzed at the single-cell level. Since topoisomerase I is a distinct autoantigen, we targeted systemic sclerosis as autoimmune disease. Decreased and increased affinities for topoisomerase I of topoisomerase I-reactive B cells led to anti-inflammatory and pro-inflammatory cytokine production associated with the inhibition and development of fibrosis, which is the major symptom of systemic sclerosis. Furthermore, inhibition of pro-inflammatory cytokine production and increased affinity of topoisomerase I-reactive B cells suppressed fibrosis. These results indicate that autoantigen-reactive B cells contribute to the disease manifestations in autoimmune disease through their antigen affinity.

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.

Super-Resolution Defocusing Nanoparticle Image Velocimetry Utilizing Spherical Aberration for Nanochannel Flows.

Understanding fluid flows and mass transport in nanospaces is becoming important with recent advances in nanofluidic analytical devices utilizing nanopores and nanochannels. In the present study, we developed a super-resolution and fast particle tracking method utilizing defocusing images with spherical aberration and demonstrated the measurement of nanochannel flow. Since the spherical aberration generates the defocusing nanoparticle image with diffraction rings, the position of fluorescent nanoparticles was determined from the radius of the diffraction ring. Effects of components of an optical system on the diffraction ring of the defocusing image were investigated and optimized to achieve the spatial resolution exceeding the optical diffraction limit. We found that there is an optimal magnitude of spherical aberration to enhance the spatial resolution. Furthermore, we confirmed that nanoparticles with diameters in the order of 101 nm, which is much smaller than the light wavelength, do not affect the defocusing images and the spatial resolution because such nanoparticles can be regarded as point light sources. At optimized conditions, we achieved a spatial resolution of 19 nm and a temporal resolution of 160 µs, which are sufficient for the nanochannel flow measurements. We succeeded in the measurement of pressure-driven flow in a nanochannel with a depth of 370 nm using 67 nm fluorescent nanoparticles. The measured nanoparticle velocities exhibited a parabolic flow profile with a slip velocity even at the hydrophilic glass surface but with an average velocity similar to the Hagen-Poiseuille law. The method will accelerate researches in the nanofluidics and other related fields.

Stable Formation of Aqueous/Organic Parallel Two-phase Flow in Nanochannels with Partial Surface Modification.

In microfluidics, various chemical processes can be integrated utilizing parallel multiphase flows. Our group has extended this research to nanofluidics, and recently performed the extraction of lipids using parallel two-phase flow in nanochannels. Although this was achieved in surface-modified nanochannels, a stable condition of parallel two-phase flow remains unknown due to difficulties in device fabrication, for a suitable method of bonding surface-modified substrates is lacking. Therefore, research on parallel two-phase flow in nanochannels has been limited. Herein, a new bonding method which improves the wash process for the substrates and increases the bonding rate to ∼100% is described. The conditions to achieve parallel organic/aqueous two-phase flow were then studied. It was revealed that in nanochannels, higher capillary numbers for the organic phase flow were required compared to that in microchannels. The newly developed fabrication process and flow regimes will contribute to realize integrated nanofluidic devices capable of analyzing single molecules/cells.

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.

Advances in Nanofluidics.

Recently, a new frontier in fluid science and engineering at the 1 to 1000 nm scale, called nanofluidics, has developed and provided new methodologies and applications to the fields of chemistry, biology, material sciences, bioengineering, medicine, drug discovery, energy, and environmental engineering [...].

Advanced Top-Down Fabrication for a Fused Silica Nanofluidic Device.

Nanofluidics have recently attracted significant attention with regard to the development of new functionalities and applications, and producing new functional devices utilizing nanofluidics will require the fabrication of nanochannels. Fused silica nanofluidic devices fabricated by top-down methods are a promising approach to realizing this goal. Our group previously demonstrated the analysis of a living single cell using such a device, incorporating nanochannels having different sizes (102-103 nm) and with branched and confluent structures and surface patterning. However, fabrication of geometrically-controlled nanochannels on the 101 nm size scale by top-down methods on a fused silica substrate, and the fabrication of micro-nano interfaces on a single substrate, remain challenging. In the present study, the smallest-ever square nanochannels (with a size of 50 nm) were fabricated on fused silica substrates by optimizing the electron beam exposure time, and the absence of channel breaks was confirmed by streaming current measurements. In addition, micro-nano interfaces between 103 nm nanochannels and 101 µm microchannels were fabricated on a single substrate by controlling the hydrophobicity of the nanochannel surfaces. A micro-nano interface for a single cell analysis device, in which a nanochannel was connected to a 101 µm single cell chamber, was also fabricated. These new fabrication procedures are expected to advance the basic technologies employed in the field of nanofluidics.

A Simple Low-Temperature Glass Bonding Process with Surface Activation by Oxygen Plasma for Micro/Nanofluidic Devices.

The bonding of glass substrates is necessary when constructing micro/nanofluidic devices for sealing micro- and nanochannels. Recently, a low-temperature glass bonding method utilizing surface activation with plasma was developed to realize micro/nanofluidic devices for various applications, but it still has issues for general use. Here, we propose a simple process of low-temperature glass bonding utilizing typical facilities available in clean rooms and applied it to the fabrication of micro/nanofluidic devices made of different glasses. In the process, the substrate surface was activated with oxygen plasma, and the glass substrates were placed in contact in a class ISO 5 clean room. The pre-bonded substrates were heated for annealing. We found an optimal concentration of oxygen plasma and achieved a bonding energy of 0.33-0.48 J/m2 in fused-silica/fused-silica glass bonding. The process was applied to the bonding of fused-silica glass and borosilicate glass, which is generally used in optical microscopy, and revealed higher bonding energy than fused-silica/fused-silica glass bonding. An annealing temperature lower than 200 °C was necessary to avoid crack generation by thermal stress due to the different thermal properties of the glasses. A fabricated micro/nanofluidic device exhibited a pressure resistance higher than 600 kPa. This work will contribute to the advancement of micro/nanofluidics.

Lipid Bilayer-Modified Nanofluidic Channels of Sizes with Hundreds of Nanometers for Characterization of Confined Water and Molecular/Ion Transport.

Water inside and between cells with dimensions on the order of 101-103 nm such as synaptic clefts and mitochondria is thought to be important to biological functions, such as signal transmissions and energy production. However, the characterization of water in such spaces has been difficult owing to the small size and complexity of cellular environments. To this end, we proposed and fabricated a biomimetic nanospace exploiting nanofluidic channels with defined dimensions of hundreds of nanometers and controlled environments. A method of modifying a glass nanochannel with a unilamellar lipid bilayer was developed. We revealed that 2.1-5.6 times higher viscosity of water arises in a 200 nm sized biomimetic nanospace by interactions between water molecules and the lipid bilayer surface and significantly affects the molecular/ion transport that is required for the biological functions. The proposed method provides both a technical breakthrough and new findings to the fields of physical chemistry and biology.

Femtoliter Volumetric Pipette and Flask Utilizing Nanofluidics.

Microfluidics has achieved integration of analytical processes in microspaces and realized miniaturized analyses in fields such as chemistry and biology. We have proposed a general concept of integration and extended this concept to the 10-1000 nm scale exploring ultimate analytical performances (e.g. immunoassay of a single-protein molecule). However, a sampling method is still challenging for nanofluidics despite its importance in analytical chemistry. In this study, we developed a femtoliter (fL) sampling method for volume measurement and sample transport. Traditionally, sampling has been performed using a volumetric pipette and flask. In this research, a nanofluidic device consisting of a femtoliter volumetric pipette and flask was fabricated on glass substrates. Since gravity, which is exploited in bulk fluidic operations, becomes less dominant than surface effects on the nanometer scale, fluidic operation of the femtoliter sampling was designed utilizing surface tension and air pressure control. The working principle of an 11 fL volumetric pipette and a 50 fL flask, which were connected by a nanochannel, was verified. It was found that evaporation of the sample solution by air flow was a significant source of error because of the ultra-small volumes being processed. Thus, the evaporation issue was solved by suppressing the air flow. As a result, the volumetric measurement error was decreased to ±0.06 fL (CV 0.6%), which is sufficiently low for use in nanofluidic analytical applications. This study will present a fundamental technology for the development of novel analytical methods for femtoliter volume samples such as single molecule analyses.

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.

Enzyme-linked immunosorbent assay utilizing thin-layered microfluidics.

A rapid and sensitive enzyme-linked immunosorbent assay (ELISA) is required for on-site clinical diagnosis. Previously, a microfluidic ELISA in which antibody-immobilized beads are packed in a microchannel for a high surface-to-volume (S/V) ratio was developed, but utilizing beads led to complicated fluidic operation. Recently, we have reported nanofluidic ELISA that utilizes antibody-immobilized glass nanochannels (102-103 nm) to achieve a high S/V ratio without beads, enabling even single-molecule detection, but it is not applicable to clinical diagnosis owing to its fL sample volume, much smaller than the nL-µL sample volume in clinical diagnosis. Here, we propose an antibody-immobilized, thin-layered microfluidic channel as a novel platform. Based on the method of nanofluidic ELISA, the channel width was expanded from 103 nm to 100 mm to expand the volume of the reaction field to 102 nL, while the channel depth (103 nm) was maintained to retain the high S/V ratio. A device design which incorporates a taper-shaped interface between the thin-layered channel and the microchannel for sample injection was proposed, and the uniform introduction of the sample into the high-aspect-ratio (width/depth ∼ 200) channel was experimentally confirmed. For the proof of concept, a thin-layered ELISA device with the same S/V ratio as the bead-based ELISA format was designed and fabricated. By measuring a standard C-reactive protein solution, the working principle was verified. The limit of detection was 34 ng mL-1, which was comparable to that of bead-based ELISA. We believe that the thin-layered ELISA can contribute to medicine and biology as a novel platform for sensitive and rapid ELISA.

Parallel multiphase nanofluidics utilizing nanochannels with partial hydrophobic surface modification and application to femtoliter solvent extraction.

In the field of microfluidics, utilizing parallel multiphase flows with immiscible liquid/liquid or gas/liquid interfaces along a microchannel has achieved the integration of various chemical processes for analyses and syntheses. Recently, our group has developed nanofluidics that exploits 100 nm nanochannels to realize ultra-small (aL to fL scale) and highly efficient chemical operations. Novel applications such as single-molecule analyses and single-cell omics are anticipated. However, the formation of parallel multiphase flows in a nanochannel remains challenging. To this end, here we developed a novel method for nanoscale partial hydrophobic surface modification of a nanochannel utilizing a focused ion beam. Hydrophobic and hydrophilic areas could be patterned beside one another even in a 60 nm glass nanochannel. Because this patterning maintained the liquid/liquid interface in the nanochannel based on the difference in wettability, stable aqueous/organic parallel two-phase flow in a 40 fL nanochannel was realized for the first time. Utilizing this flow, nanoscale unit operations involving phase confluence, extraction and phase separation were integrated to demonstrate solvent extraction of a lipid according to the Bligh-Dyer method, which is a broadly used pretreatment process in lipidomics. We accomplished the separation of a lipid and an amino acid in a sample volume of 4 fL (250 times smaller than the pL volume of a single cell) with a processing time of 1 ms (10 000 times faster than that in a microchannel). This study therefore provides a technological breakthrough that advances the field of nanofluidics to allow multiphase chemical processing at fL volumes.

Femtoliter nanofluidic valve utilizing glass deformation.

In the field of micro/nanofluidics, the channel open/close valves are among the most important technologies for switching and partitioning actions and integration of various operations into fluidic circuits. While several types of valves have been developed in microfluidics, few are capable in nanofluidics. In this study, we proposed a femtoliter (fL) volume nanochannel open/close valve fabricated in glass substrates. The valve consists of a shallow, circular and stepped-bottom valve chamber connected to nanochannels and an actuator. Even with tiny deformation occurring at the nanolevel in glass, an open/closed state of a nanochannel (10-1000 nm) can be achieved. We designed a fL-valve based on an analytical material deformation model, and developed a valve fabrication process. We then verified the open/closed state of the valve using a 308 fL-valve chamber with a four-stepped nanostructure fitting an arc-shape of deflected glass, confirmed its stability and durability over 50 open/close operations, and succeeded in stopping/flowing an aqueous solution at 209 fL s-1 under a 100 kPa pressure in a 900 nm nanochannel with a fast response of ∼0.65 s. A leak flow from the closed valve was sufficiently small even at a 490 kPa pressure-driven flow. Since the developed fL-valve can be applied to various nanofluidic devices made of glass and other rigid materials such as plastic, it is expected that this work will contribute significantly to the development of novel integrated micro/nanofluidics chemical systems for use in various applications, such as single cell/single molecule analysis.

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.