A numerical model was established for photothermal detection of non-fluorescent molecules confined in micro/nanofluidic channels. Recently, nanofluidics has been accepted as a methodology to analyze ...the behavior of single molecules. The extremely high surface-to-volume ratio of the nanospace is also promising for biochemical reactors. However, detection of the solute molecules confined in the nanospace is still difficult unless the molecules are fluorescently labeled. To date, our group has studied photothermal spectroscopy, which can detect non-fluorescent molecules using thermal relaxation, and developed photothermal optical phase shift (POPS) detection. The POPS detector has realized a detection limit of 30 protein molecules, but heat transfer from the sample solution to the glass nanochannel is a barrier to achieve single-molecule sensitivity. In this study, a finite element method (FEM) analysis was performed to simulate the photothermal effect and heat transfer in the nanochannels. The POPS signal calculated from the temperature distribution was compared with experimental values to validate the numerical model. The influences of the detection parameters, including channel depth, modulation frequency, and misalignment of laser beams, are all discussed for improvement of the sensitivity.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Single molecule analysis is desired in many areas that require the analysis of ultra-small volume and/or extremely low concentration samples (e.g., single-cell biology, medicine diagnosis, virus ...detection, etc.). Due to the ultra-small volume or concentration, the sample contains only single or countable analyte molecules. Thus, specific single molecules should be precisely processed and detected for analysis. However, except nucleic acids, most molecules are difficult to amplify, and a new analytical methodology for specific single molecules is thus essential. For this, efficient chemical processing and detection, which are important analytical elements, should be developed. Here, we report a single-molecule ELISA (enzyme-linked immunosorbent assay) device utilizing micro/nanofluidic technology. Both chemical processing and detection were integrated into an ultra-small space (10
nm in size), and the integration allowed precise processing (∼100% capture) and detection of a specific single molecule (protein) for the first time. This new concept and enabling technology represent a significant innovation in analytical chemistry and will have a large impact on general biology and medicine.
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.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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 (10
2
-10
3
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 10
3
nm to 10
0
mm to expand the volume of the reaction field to 10
2
nL, while the channel depth (10
3
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.
An antibody-immobilized thin-layered glass microfluidic channel with a high surface-to-volume ratio was developed for rapid and sensitive enzyme-linked immunosorbent assay.
The expansion of microfluidics research to nanofluidics requires absolutely sensitive and universal detection methods. Photothermal detection, which utilizes optical absorption and nonradiative ...relaxation, is promising for the sensitive detection of nonlabeled biomolecules in nanofluidic channels. We have previously developed a photothermal optical phase shift (POPS) detection method to detect nonfluorescent molecules sensitively, while a rapid decrease of the sensitivity in nanochannels and the introduction of an ultraviolet (UV) excitation system were issues to be addressed. In the present study, our primary aim is to characterize the POPS signal in terms of the thermo-optical properties and quantitatively evaluate the causes for the decrease in sensitivity. The UV excitation system is then introduced into the POPS detector to realize the sensitive detection of nonlabeled biomolecules. The UV-POPS detection system is designed and constructed from scratch based on a symmetric microscope. The results of simulations and experiments reveal that the sensitivity decreases due to a reduction of the detection volume, dissipation of the heat, and cancellation of the changes in the refractive indices. Finally, determination of the concentration of a nonlabeled protein (bovine serum albumin) is performed in a very thin 900 nm deep nanochannel. As a result, the limit of detection (LOD) is 2.3 μM (600 molecules in the 440 attoliter detection volume), which is as low as that previously obtained for our visible POPS detector. UV-POPS detection is thus expected be a powerful technique for the study of biomolecules, including DNAs and proteins confined in nanofluidic channels.
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IJS, KILJ, NUK, PNG, UL, UM
•The extended-nano (100nm scale) fluidic channels were modified with ODS (C18).•The reversed phase HPLC was realized in extended-nano space.•The six amino acids derivatized with NBD-F were separated ...on extended-nano space.•The injection volume of 30fL and plate height of 3μm was achieved.•This extended-nano space chromatography can be applied for single cell analysis.
In this work we used reversed-phase chromatography in extended-nano channels to separate amino acids. A hydrophobic surface modification of extended-nano channels was established. A sample mixture of fluorescein and sulforhodamine B (0.5 and 0.05mM respectively) was used for the demonstration of a reversed-phase separation mode. A small amount of sample band (30fL) was injected into the separation channel, and two compounds were successfully separated. The maximum theoretical plate number of sulforhodamine B was 300,000plates/m. Two sets of 3 amino acids (3.75mM each) were separated using 0.01M citrate buffer (pH 5.5) with 0.01M sodium perchlorate and 12 and 25% of acetonitrile as a mobile phase. A successful separation (320,000plates/m with plate height of 3.2μm for serine) was accomplished.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Nanofluidics, a discipline of science and engineering of fluids confined to structures at the 1–1000 nm scale, has experienced significant growth over the past decade. Nanofluidics have offered ...fascinating platforms for chemical and biological analyses by exploiting the unique characteristics of liquids and molecules confined in nanospaces; however, the difficulty to detect molecules in extremely small spaces hampers the practical applications of nanofluidic devices. Laser-induced fluorescence microscopy with single-molecule sensitivity has been so far a major detection method in nanofluidics, but issues arising from labeling and photobleaching limit its application. Recently, numerous label-free detection methods have been developed to identify and determine the number of molecules, as well as provide chemical, conformational, and kinetic information of molecules. This review focuses on label-free detection techniques designed for nanofluidics; these techniques are divided into two groups: optical and electrical/electrochemical detection methods. In this review, we discuss on the developed nanofluidic device architectures, elucidate the mechanisms by which the utilization of nanofluidics in manipulating molecules and controlling light–matter interactions enhances the capabilities of biological and chemical analyses, and highlight new research directions in the field of detections in nanofluidics.
Sensitive detection and quantification of individual plasmonic nanoparticles is critical in a range of applications in the biological, nanomaterials, and analytical sciences. Although a wide range of ...techniques can be applied to the analysis of immobilized particles, high-throughput analysis of nanoscale species in flow is surprisingly underdeveloped. To address this shortcoming, we present an ultrasensitive, background-free technique based on the photothermal effect and termed differential detection photothermal interferometry (DDPI). We show, both theoretically and experimentally, that DDPI can specifically extract either the phase or amplitude of a photothermal signal. We then quantitatively detect 10 and 20 nm diameter gold nanoparticles at femtomolar concentrations and at linear flow speeds of 10 mm/s. In the case of 50 nm gold particles, we operate at an even higher linear flow speed of 100 mm/s, corresponding to an analyzed volume of more than 1 nL/s. This allows quantification of particle content at attomolar to femtomolar concentrations and counting rates between 0.1 and 400 particles per second. Finally, we confirm that the signal follows the size-dependent variations predicted by Mie theory.
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IJS, KILJ, NUK, PNG, UL, UM
The separation and sensitive detection of nonfluorescent molecules at the femtoliter (fL) scale has been achieved for the first time in a nanofluidic channel. Smaller sample volumes and higher ...separation efficiencies have been significant targets for liquid chromatography for many years. However, the use of packed columns hindered further miniaturization and improvement of separation efficiency. Our group recently developed a novel chromatographic method using an open nanofluidic channel to realize attoliter sample injection and a separation efficiency of several million plates per m. However, because of the extremely small optical path length, this detection method was limited to fluorescent molecules. Herein, we describe the combination of nanofluidic chromatography with differential interference contrast thermal lens microscopy (DIC-TLM), a sensitive detection method for nonfluorescent molecules developed by our group that has the ability to detect 0.61 zmol (370 molecules) with an optical path length of 350 nm. As a result, separation of a 21 fL sample containing 250 zmol was possible at the limit of detection (LOD).
Liquid chromatography using a nanofluidic chip and DIC-TLM realized separation and detection of a 21 fL, 0.61 zmol nonfluorescent sample.
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