Nanofluidic Detection Platform for Simultaneous Light Absorption and Scattering Measurement of Individual Nanoparticles in Flow.

Characterization and quantification of plasmonic nanoparticles at the single particle level have become increasingly important with the advancements in nanotechnology and their application to various biological analyses including diagnostics, photothermal therapy, and immunoassays. While various nanoparticle detection methodologies have been developed and widely used, simultaneous measurement of light absorption and scattering from individual plasmonic nanoparticles in flow is still challenging. Herein, we describe a novel nanofluidic detection platform that enables simultaneous measurement of absorption and scattering signals from individual nanoparticles within a nanochannel. Our detection platform utilized optical diffraction phenomena by a single nanochannel as both a readout signal for photothermal detection and a reference light for interferometric scattering detection. Through the elucidation of the frequency effect on the detection performance and optimization of experimental conditions, we achieved the classification of gold and silver nanoparticles with a diameter of 20-60 nm at an average accuracy score of 82.6 ± 2.1% by measured data sets of absorption and scattering signals. Furthermore, we demonstrated the concentration determination of plasmonic nanoparticle mixtures using a trained Support vector machine (SVM) classifier. Our simple yet sensitive nanofluidic detection platform will be a valuable tool for the analysis of nanoparticles and their applications to chemical and biological assays.

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.

Proton diffusion and hydrolysis enzymatic reaction in 100 nm scale biomimetic nanochannels.

Liquids in 10-100 nm spaces are expected to play an important role in biological systems. However, the liquid properties and their influence on biological activity have been obscured due to the difficulty in nanoscale measurements, either in vivo or in vitro. In this study, an in vitro analytical platform for biological systems is established. The nanochannels were modified with lipid bilayers, thereby serving as a model for biological confinement, e.g., the intercellular or intracellular space. As a representative property, the proton diffusion coefficient was measured by a nanofluidic circuit using fluorescein as a pH probe. It was verified that proton conduction was enhanced for channel widths less than 330 nm. A proton-related enzymatic reaction, the hydrolysis reaction, was also investigated, and a large confinement effect was observed.

Correction: Integration of sequential analytical processes into sub-100 nm channels: volumetric sampling, chromatographic separation, and label-free molecule detection.

Correction for 'Integration of sequential analytical processes into sub-100 nm channels: volumetric sampling, chromatographic separation, and label-free molecule detection' by Yoshiyuki Tsuyama et al., Nanoscale, 2021, 13, 8855-8863, https://doi.org/10.1039/D0NR08385B.

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.

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.

Metal-Free Fabrication of Fused Silica Extended Nanofluidic Channel to Remove Artifacts in Chemical Analysis.

In microfluidics, especially in nanofluidics, nanochannels with functionalized surfaces have recently attracted attention for use as a new tool for the investigation of chemical reaction fields. Molecules handled in the reaction field can reach the single-molecule level due to the small size of the nanochannel. In such surroundings, contamination of the channel surface should be removed at the single-molecule level. In this study, it was assumed that metal materials could contaminate the nanochannels during the fabrication processes; therefore, we aimed to develop metal-free fabrication processes. Fused silica channels 1000 nm-deep were conventionally fabricated using a chromium mask. Instead of chromium, electron beam resists more than 1000 nm thick were used and the lithography conditions were optimized. From the results of optimization, channels with 1000 nm scale width and depth were fabricated on fused silica substrates without the use of a chromium mask. In nanofluidic experiments, an oxidation reaction was observed in a device fabricated by conventional fabrication processes using a chromium mask. It was found that Cr6+ remained on the channel surfaces and reacted with chemicals in the liquid phase in the extended nanochannels; this effect occurred at least to the micromolar level. In contrast, the device fabricated with metal-free processes was free of artifacts induced by the presence of chromium. The developed fabrication processes and results of this study will be a significant contribution to the fundamental technologies employed in the fields of microfluidics and nanofluidics.

Integration of sequential analytical processes into sub-100 nm channels: volumetric sampling, chromatographic separation, and label-free molecule detection.

The progress of nanotechnology has developed nanofluidic devices utilizing nanochannels with a width and/or depth of sub-100 nm (101 nm channels), and several experiments have been implemented in ultra-small spaces comparable to DNAs and proteins. However, current experiments utilizing 101 nm channels focus on a single function or operation; integration of multiple analytical operations into 101 nm channels using nanofluidic circuits and fluidic control has yet to be realized despite the advantage of nanochannels. Herein, we report the establishment of a label-free molecule detection method for 101 nm channels and demonstration of sequential analytical processes using integrated nanofluidic devices. Our absorption-based detection method called photothermal optical diffraction (POD) enables non-invasive label-free molecule detection in 101 nm channels for the first time, and the limit of detection (LOD) of 1.8 µM is achieved in 70 nm wide and deep nanochannels, which corresponds to 7.5 molecules in the detection volume of 7 aL. As a demonstration of sampling in 101 nm channels, aL-fL volumetric sampling is performed using 90 nm deep cross-shaped nanochannels and pressure-driven fluidic control from three directions. Finally, the POD and volumetric sampling are combined with nanochannel chromatography, and separation analysis in 101 nm channels is demonstrated. The experimental results reported in this paper will contribute to the advances in 101 nm fluidic devices which have the potential to provide a novel platform for chemical/biological analyses.

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.

Diffraction-based label-free photothermal detector for separation analyses in a nanocapillary.

Miniaturization of column diameter in liquid chromatography is one of the major trends in separation sciences toward single-cell proteomics and metabolomics. Micro/nanoscale open tubular (OT) capillaries are promising tools for efficient separation analyses of the ultra-small volume of samples. However, highly sensitive and label-free on-column detection is still challenging for such ultra-small capillaries. In this study, we developed a photothermal detector using optical diffraction phenomena by a single nanocapillary. Our detection method realized concentration determination of unlabeled sample solutions in a nanocapillary with 460 nm inner diameter. The calculated limit of detection was 0.12 µM, which corresponds to 16 molecules in a detection volume of 0.23 fL. Furthermore, normal-phase chromatography was performed on a 12 cm long nanocapillary, and femtoliter sample injection, efficient separation, and label-free detection of dye molecules were demonstrated. Our photothermal detector will be widely used as a universal tool for chemical/biological analyses using capillaries with micro/nanoscale diameters.

Isotope Effect in the Liquid Properties of Water Confined in 100 nm Nanofluidic Channels.

Liquids confined in 10-100 nm spaces show different liquid properties from those in the bulk. Proton transfer plays an essential role in liquid properties. The Grotthuss mechanism, in which charge transfer occurs among neighboring water molecules, is considered to be dominant in bulk water. However, the rotational motion and proton transfer kinetics have not been studied well, which makes further analysis difficult. In this study, an isotope effect was used to study the kinetic effect of rotational motion and proton hopping processes by measurement of the viscosity, proton diffusion coefficient, and the proton hopping activation energy. As a result, a significant isotope effect was observed. These results indicate that the rotational motion is not significant, and the decrease of the proton hopping activation energy enhances the apparent proton diffusion coefficient.

Concentration Determination at a Countable Molecular Level in Nanofluidics by Solvent-Enhanced Photothermal Optical Diffraction.

Nanofluidic devices have become a powerful tool for extremely precise analyses at a single-molecule/nanoparticle level. However, a simple and sensitive molecular detection method is essential for nanofluidic devices because of ultrasmall volume (fL-aL). One such technology is photothermal spectroscopy (PTS), which utilizes light absorption and thermal relaxation by target molecules. Recently, we developed a photothermal optical diffraction (POD) detection method as PTS for nanofluidic devices. However, the detectable concentration range was in the order of µM (102 to 104 molecules), and further improvement in detection performance is strongly required. Here, we demonstrate solvent-enhanced POD with optimized experimental conditions and show its capability of concentration determination of nonfluorescent molecules in nanochannels at a countable molecular level. A relationship between the POD signal and thermal/optical properties of solvents is elucidated. We estimate the diffraction factor and photothermal factor of the solvent enhancement effect by thermal simulations and theoretical calculations. Experimental results show good agreement with the prediction, and the detection performance of the POD is successfully improved. At the optimized condition, we demonstrate the concentration determination with the limit of detection of 75 nM, which corresponds to an average of 10 molecules in a detection volume of 0.23 fL. Our sensitive nonfluorescent molecule detection method will be applied to a wide range of chemical/biological analyses utilizing 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.

Nanochannel chromatography and photothermal optical diffraction: Femtoliter sample separation and label-free zeptomole detection.

Column miniaturization of liquid chromatography is a major trend in separation sciences with the advent of single-cell proteomics and metabolomics. Nanochannel chromatography is one of the promising tools for single-cell analyses because it provides ultra-small sample volume and high separation efficiency. However, non-fluorescent molecular detection in such small channels is still quite difficult due to fL-aL sample volume, which hinders further miniaturization of nanochannels. In this study, we overcame the size limitation of nanochannel chromatography by our label-free molecular detection method: photothermal optical diffraction (POD), which utilizes the photothermal effect of analytes and optical diffraction by nanochannel. The combination of the nanochannel chromatography and the POD enables 1.8 fL sample separation and label-free molecule detection in nanochannels with 800 nm width and 300 nm depth at the optimized experimental conditions. The limit of detection is 5.4 zmol (3300 molecules), approximately 50 times lower than the conventional label-free detection method. Furthermore, the theoretical plate number is calculated to be 105 plates/m, and the separation performance is discussed. Our label-free detection method will be widely used as a universal detector for nanochannel chromatography.

Ultrasensitive detection of nonlabelled bovine serum albumin using photothermal optical phase shift detection with UV excitation.

Ultrasensitive detection of nonlabelled bovine serum albumin is performed in micro/nanofluidic chips using a photothermal optical phase shift (POPS) detection system. Currently, micro- and nanofluidics allow the analysis of various single cells, and their targets of interest are shifting from nucleic acids to proteins. Previously, our group developed photothermal detection techniques for the sensitive detection of nonfluorescent molecules. For example, we developed a thermal lens microscope (TLM) with ultrahigh sensitivity at the single-molecule level and a POPS detector that is applicable to nanochannels smaller than the wavelength of light. The POPS detector also realized the detection of nonlabelled proteins in nanochannels, although its detection sensitivity is less than that of the TLM in microchannels due to insufficient background light reduction. To overcome this problem, we developed a new POPS detector using relay optics for further reduction of the background light. In addition, heat transfer from the sample solution to the nanochannel wall was thoroughly investigated to achieve ultrahigh sensitivity. The limit of detection (LOD) obtained with the new POPS detector is 30 molecules in 1.0 fL. Considering this LOD, the performance of the new POPS detector is comparable with that of the TLM. Owing to the applicability of the POPS detector for sensitive detection even in nanochannels or single-µm channels, which cannot be realized with the TLM, combinations of the POPS detector and separation techniques employing unique nanochannel properties will contribute to advances in single-cell proteomics in the future.

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.

Detection and Characterization of Individual Nanoparticles in a Liquid by Photothermal Optical Diffraction and Nanofluidics.

Detection and characterization of individual nanoparticles less than 100 nm are important for semiconductor manufacturing, environmental monitoring, biomedical diagnostics, and drug delivery. Photothermal spectroscopy is a light absorptiometry and promising method for detection and characterization because of its high sensitivity and selectivity compared with light scattering or electrical detection methods. However, the characterization of individual nanoparticles in liquids is still challenging for conventional photothermal detection methods. Here, we report a method for the ultrasensitive detection and accurate characterization of individual nanoparticles in liquids by photothermal optical diffraction, which utilizes enhancement of optical diffraction by a nanochannel after light absorption and heat generation of individual nanoparticles in the channel. Our method realized individual 20 nm Au nanoparticle detection with almost 100% detection efficiency by utilizing nanochannels, leading to concentration determination without a calibration curve. Furthermore, we measured individual nanoparticle size and discriminated 20 and 40 nm Au nanoparticles from their photothermal signals. Our photothermal-based nanoparticle detection method in nanochannels has a potential for a wide range of applications such as on-site evaluation of synthesized plasmonic nanoparticles and drug delivery particles.

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.