ABSTRACT
Miniaturized systems have attracted much attention with the recent advances in microfluidics and nanofluidics. From the capillary electrophoresis, the development of glass-based microfluidic and nanofluidic technologies has supported advances in microfluidics and nanofluidics. Most microfluidic systems, especially nanofluidic systems, are still simple, such as systems constructed with simple straight nanochannels and bulk-scale electrodes. One of the bottlenecks to the development of more complicated and sophisticated systems is to develop the locally integrated nano-electrodes. However, there are still issues with integrating nano-electrodes into nanofluidic devices because it is difficult to fit the nano-electrode size into a nanofluidic channel at the nanometer level. In this study, we propose a new method for the fabrication of local nano-electrodes in nanofluidic devices with nanofluidic and nano-electrochemistry-based experiments. An electroplating solution was introduced to a nanochannel with control of the flow and the electroplating reaction, by which nano-electrodes were successfully fabricated. In addition, a nanofluidic device was available for nanofluidic experiments with the application of 200 kPa. This method can be applied to any electroplating material such as gold and copper. The local nano-electrode will make a significant contribution to the development of more complicated and sophisticated nanofluidic electrophoresis systems and to local electric detection methods for various nanofluidic devices.
ABSTRACT
Fused-silica glass, as a desirable material with rigidity, biological inertness, and favorable light transmission for nanofluidic devices, should be assembled via low-temperature bonding technology to hermetically seal channels for stable liquid manipulation in extended-nano (101-103 nm) space. Confronted with the predicament of localized functionalization of nanofluidic applications (e.g. DNA microarray) with temperature-sensitive structures, the room-temperature direct bonding of glass chips to achieve modification of channels prior to bonding offers a considerably attractive solution to avoid component denaturation during the conventional post-bonding heating process. Therefore, we developed a nano-structure friendly and technically convenient room-temperature (RT, 25 °C) glass-glass direct bonding technology using polytetrafluoroethylene (PTFE)-assisted plasma modification without a requirement for special equipment. Unlike the establishment of chemical functionalities relying on immersion in potent but dangerous chemicals like HF, the fluorine radicals (F*) from PTFE pieces with superior chemical inertness were introduced on glass surfaces via O2 plasma sputtering and constructed fluorinated silicon oxides on the glass surfaces effectively, eliminating the significant etching effect of HF to protect fine nanostructures. Very strong bonding was obtained at RT with no heating and the high-pressure resistant glass-glass interfaces were evaluated under high-pressure-driven flow conditions up to 2 MPa based on a two-channel liquid introduction system. Moreover, the favorable optical transmittance of the fluorinated bonding interface demonstrated a capacity for high-resolution optical detection or liquid sensing.
ABSTRACT
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.
ABSTRACT
The lifetime of an enzyme is critical to prevent system failure and optimize maintenance schedules in biological and analytical chemistry. The lifetime metrics of an enzyme can be evaluated from enzyme activity in terms of catalytic cycles per enzyme at various storage times. Trypsin, which is a gold-standard enzyme in proteomics, has been known to decrease activity due to self-digestion. To improve the activity of trypsin, enzyme reactors have developed by immobilizing in micro and nanospace. However, an evaluation method for the catalytic cycle has not been established due to major issues such as nonuniform space, unstable liquid transport, and self-digestion during immobilization in conventional work. To solve these issues, we have previously developed an ultra-fast enzyme reactor with a well-defined nanofabrication method, stable liquid transport, and partial enzyme modification. Here, we aimed to investigate catalytic cycles in a nanochannel. To extend enzyme lifetime efficiently, we have evaluated the optimal immobilization process and catalytic cycles of trypsin. As a result, immobilized enzyme densities by the trypsinogen immobilization process were increased at all concentrations compared to the trypsin immobilization process. To evaluate the lifetime of trypsin, the immobilized enzyme densities and activities were almost the same before and after 72 h of enzyme storage, and the calculated catalytic cycles were 1740. These results indicated that self-digestion of the immobilized enzyme was highly suppressed. Consequently, the reaction efficiency has been evaluated depending on the catalytic cycles from the substrate for the first time, while preventing self-digestion by trypsin.
Subject(s)
Enzymes, Immobilized , Proteomics , Enzymes, Immobilized/metabolism , Trypsin/metabolism , Proteomics/methods , Catalysis , Bioreactors , Enzyme StabilityABSTRACT
We developed a process for enzyme-linked immunosorbent assay on a glass microchip via the use of a thin-layered microfluidic channel. This channel possesses a high aspect ratio (width/depth â¼200) and has an antibody layer immobilized directly on the channel surface. A depth of several microns and an excessive width and length (mm scale) of the channel provide a large-volume capacity (102 nL) and maximum capture efficiency of the analyte for a high level of detection sensitivity (102 pg mL-1). The developed reusable immunosensor has demonstrated high-performance characteristics by requiring less than 50 µL of sample and providing analysis in less than 25 min. This new method could impact the development of point-of-care devices for biomedical applications.
Subject(s)
Biosensing Techniques , Microfluidics , Microfluidics/methods , Immunoassay , Enzyme-Linked Immunosorbent Assay/methods , ProteinsABSTRACT
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.
Subject(s)
Enzyme-Linked Immunosorbent Assay , Microfluidic Analytical Techniques , Enzyme-Linked Immunosorbent Assay/methods , Interleukin-6/chemistry , Microfluidic Analytical Techniques/methods , Nanotechnology/methodsABSTRACT
Nanostructures can realize highly efficient reactions due to their structural advantages. However, the mechanism of accelerating enzyme reactions in a nanospace is still unknown from a kinetic perspective because it is difficult to control a well-defined nanospace, enzyme density, and reaction time. Here, we investigated kinetic parameters of an immobilized enzyme in micro- and nanochannels using nanofabrication, partial enzyme patterning, fluidic control, and a high sensitivity detection system. Devices with channel depths of 300 nm, 4.4 µm, and 13.6 µm were fabricated. Kinetic parameters were determined by the Michaelis-Menten model. Compared to the bulk reaction, all kcats for immobilized enzyme reactors were decreased, although the kcats were approximately the same for the immobilized enzyme reactors of different depths. An ultrafast enzyme reaction could overcome the drawback due to immobilization by an increase of the apparent [E]0 due to the decreased channel depth.
Subject(s)
Enzymes, Immobilized , Kinetics , Enzymes, Immobilized/chemistryABSTRACT
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.
ABSTRACT
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.
Subject(s)
Proteins , Spectrometry, Mass, Electrospray Ionization , Mechanical Phenomena , Spectrometry, Mass, Electrospray Ionization/methodsABSTRACT
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.
Subject(s)
Nanotechnology , Water , RheologyABSTRACT
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.
Subject(s)
Proteome , Proteomics , Mass Spectrometry , Proteolysis , Proteomics/methods , Single-Cell AnalysisABSTRACT
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.
Subject(s)
B-Lymphocytes/immunology , DNA Topoisomerases, Type I , Scleroderma, Systemic/immunology , Scleroderma, Systemic/pathology , Adult , Animals , Autoantigens , B-Lymphocytes/pathology , Cytokines/metabolism , Disease Models, Animal , Female , Fibrosis/immunology , Fibrosis/pathology , Humans , Male , Mice , Middle AgedABSTRACT
In nanofluidics, surface control is a critical technology because nanospaces are surface-governed spaces as a consequence of their extremely high surface-to-volume ratio. Various surface patterning methods have been developed, including patterning on an open substrate and patterning using a liquid modifier in microchannels. However, the surface patterning of a closed nanochannel is difficult. In addition, the surface evaluation of closed nanochannels is difficult because of a lack of appropriate experimental tools. In this study, we verified the surface patterning of a closed nanochannel by vacuum ultraviolet (VUV) light and evaluated the surface using streaming-current measurements. First, the C18 modification of closed nanochannels was confirmed by Laplace pressure measurements. In addition, no streaming-current signal was detected for the C18-modified surface, confirming the successful modification of the nanochannel surface with C18 groups. The C18 groups were subsequently decomposed by VUV light, and the nanochannel surface became hydrophilic because of the presence of silanol groups. In streaming-current measurements, the current signals increased in amplitude with increasing VUV light irradiation time, indicating the decomposition of the C18 groups on the closed nanochannel surfaces. Finally, hydrophilic/hydrophobic patterning by VUV light was performed in a nanochannel. Capillary filling experiments confirmed the presence of a hydrophilic/hydrophobic interface. Therefore, VUV patterning in a closed nanochannel was demonstrated, and the surface of a closed nanochannel was successfully evaluated using streaming-current measurements.
ABSTRACT
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.
Subject(s)
Fibroblasts/immunology , Interleukins/genetics , Receptors, Interleukin/genetics , Scleroderma, Systemic/immunology , Th2 Cells/immunology , Adult , Aged , Animals , Antibodies, Monoclonal/pharmacology , Collagen Type I/genetics , Collagen Type I/immunology , Collagen Type I, alpha 1 Chain , Disease Models, Animal , Female , Fibroblasts/drug effects , Fibroblasts/pathology , Fibrosis , Gene Expression Regulation , Humans , Interleukin-13/genetics , Interleukin-13/immunology , Interleukin-4/genetics , Interleukin-4/immunology , Interleukin-6/genetics , Interleukin-6/immunology , Interleukins/immunology , Male , Mice , Middle Aged , Protein Isoforms/genetics , Protein Isoforms/immunology , Receptors, Interleukin/antagonists & inhibitors , Receptors, Interleukin/immunology , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/immunology , Scleroderma, Systemic/drug therapy , Scleroderma, Systemic/genetics , Scleroderma, Systemic/pathology , Skin/drug effects , Skin/immunology , Skin/pathology , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/pathology , Th2 Cells/drug effects , Th2 Cells/pathologyABSTRACT
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.
Subject(s)
Nanoparticles , RheologyABSTRACT
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.
ABSTRACT
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.
Subject(s)
B-Lymphocytes/pathology , Cell Differentiation/physiology , Lymphocyte Depletion/methods , Macrophages/pathology , Pulmonary Fibrosis/therapy , Scleroderma, Systemic/therapy , Animals , Bleomycin , Disease Models, Animal , Mice , Pulmonary Fibrosis/chemically induced , Scleroderma, Systemic/chemically induced , Scleroderma, Systemic/pathology , Skin/pathologyABSTRACT
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 â¼100% 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.
Subject(s)
Microfluidics , Nanotechnology , WaterABSTRACT
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.
ABSTRACT
Nanofluidic devices have offered us fascinating analytical platforms for chemical and bioanalysis by exploiting unique properties of liquids and molecules confined in nanospaces. The increasing interests in nanofluidic analytical devices have triggered the development of new robust and sensitive detection techniques, especially label-free ones. IR absorption spectroscopy is one of the most powerful biochemical analysis methods for identification and quantitative measurement of chemical species in the label-free and non-invasive fashion. However, the low sensitivity and the difficulties in fabrication of IR-compatible nanofluidic devices are major obstacles that restrict the applications of IR spectroscopy in nanofluidics. Here, we realized the bonding of CaF2 and SiO2 at room temperature and demonstrated an IR-compatible nanofluidic device that allowed the IR spectroscopy in a wide range of mid-IR regime. We also performed the integration of metal-insulator-metal perfect absorber metamaterials into nanofluidic devices for plasmon-enhanced infrared absorption spectroscopy with ultrahigh sensitivity. This study also shows a proof-of-concept of the multi-band absorber by combining different types of nanostructures. The results indicate the potential of implementing metamaterials in tracking several characteristic molecular vibrational modes simultaneously, making it possible to identify molecular species in mixture or complex biological entities.