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Fluorescence polarization (FP) assays are widely used to quantify biomolecules, and their combination with microfluidic devices has the potential for application in onsite analysis. However, the hydrophobic surface of polydimethylsiloxane (PDMS)-based microfluidic devices and the amphiphilicity of the blocking agents can cause the nonspecific adsorption of biomolecules, which in turn reduces the sensitivity of the FP assay. To address this, we demonstrated an FP assay with improved sensitivity in microfluidic devices using a polyethylene glycol-based surface modification to avoid the use of blocking agents. We evaluated the effectiveness of the modification in inhibiting nonspecific protein adsorption and demonstrated the improved sensitivity of the FP immunoassay (FPIA). Our study addressed the lack of sensitivity of FP assays in microfluidic devices, particularly for the quantification of low-abundance analytes.
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Combining the photoisomerization of molecules with an electrical device is important for developing optoelectronic devices. Field effect transistors (FETs) with atomically thin channels are suitable for this purpose because the FET properties respond to chemical changes in molecules. Since the photoisomerization wavelength of the switching molecules can be tuned, complex logic operations can be realized if a specific molecule is delivered to the target FET of an integrated circuit. However, conventional techniques for transferring molecules, such as drop casting and sublimation, cannot efficiently realize this goal. In this study, we fabricated a MoS2FET device combined with a microfluidic platform, wherein the MoS2channel was in contact with the flow of an azobenzene solution in isopropyl alcohol as the solvent. UV radiation (365 nm) and thermal relaxation realize the cycle of trans- and cis-azobenzene states and the switching of the substantial FET properties. This study demonstrated the feasibility of using the solution for optical switching of the MoS2-FET, which can realize quick phase changes in the molecule and the delivery of the molecule to the target FET by a microfluidic platform.
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Elucidating the link between amyloid fibril formation and liquid-liquid phase separation (LLPS) is crucial in understanding the pathologies of various intractable human diseases. However, the effect of condensed protein droplets generated by LLPS on nucleation (the initial step of amyloid formation) remains unclear because of the lack of available quantitative analysis techniques. This study aimed to develop a measurement method for the amyloid droplet nucleation rate based on image analysis. We developed a method to fix micrometer-sized droplets in gel for long-term observation of protein droplets with known droplet volumes. By combining this method with image analysis, we determined the nucleation dynamics in droplets of a prion disease model protein, Sup35NM, at the single-event level. We found that the nucleation was unexpectedly suppressed by LLPS above the critical concentration (C*) and enhanced below C*. We also revealed that the lag time in the Thioflavin T assay, a semi-quantitative parameter of amyloid nucleation rate, does not necessarily reflect nucleation tendencies in droplets. Our results suggest that LLPS can suppress amyloid nucleation, contrary to the conventional hypothesis that LLPS enhances it. We believe that the proposed quantitative analytical method will provide insights into the role of LLPS from a pathological perspective.
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Amiloide , Príons , Humanos , Amiloide/metabolismoRESUMO
Micron-sized water-in-oil droplets (microdroplets) have been used for various biochemical analyses. Many studies have been reported on immunoassays using microdroplets because of their high versatility. A selective enrichment method using spontaneous emulsification was developed as a pretreatment method for analytical systems of microdroplets. In this study, a one-step immunoassay for microdroplets using nanoparticle assembly at the interface by spontaneous emulsification is proposed. At the interface of the microdroplet, with aqueous nanoparticle dispersion, it was found that nanoparticles with diameters less than 50 nm were uniformly adsorbed to the microdroplet interface as a Pickering emulsion, whereas larger nanoparticles tended to aggregate in the bulk part of the microdroplet. Based on this phenomenon, a proof of concept of the one-step immunoassay was demonstrated using rabbit IgG as the analyte. This method is expected to be a powerful tool for trace biochemical analyses.
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Nanopartículas , Água , EmulsõesRESUMO
There is a demand for biosensors working under in vivo conditions, which requires significant device size and endurance miniaturization in solution environments. We demonstrated the detection of uric acid (UA) molecules, a marker of diseases like gout, whose continuous monitoring is required in medical diagnosis. We used a field effect transistor (FET) composed of an atomically thin transition metal dichalcogenide (TMD) channel. The sensor detection was carried out in a solution environment, for which we protected the electrodes of the source and drain from the solution. A microfluidic channel controls the solution flow that can realize evaporation-free conditions and provide an accurate concentration and precise measurement. We detected a systematic change of the drain current with the concentration of the UA in isopropyl alcohol (IPA) solvent with a detection limit of 60 nM. The sensor behavior is reversible, and the drain current returns to its original value when the channel is washed with pure solvent. The results demonstrate the feasibility of applying the MoS2-FET device to UA detection in solution, suggesting its possible use in the solution environment.
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Nowadays, the diagnosis of viral infections is receiving broad attention. We have developed a non-competitive fluorescence polarization immunoassay (NC-FPIA), which is a separation-free immunoassay, for a virus detection. H5 subtype avian influenza virus (H5-AIV) was used as a model virus for the proof of concept. The fluorescein-labeled Fab fragment that binds to H5 hemagglutinin was used for NC-FPIA. The purified H5-AIV which has H5 hemagglutinin was mixed with the fluorescein-labeled Fab fragment. After that, the degree of fluorescence polarization was measured with a portable FPIA analyzer. H5-AIV was successfully detected with an incubation time of 15 min. In addition, the portable FPIA analyzer enables performance of on-site NC-FPIA with a sample volume of 20 µL or less. This is the first research of detecting a virus particle by FPIA. This NC-FPIA can be applied to rapid on-site diagnosis of various viruses.
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Imunoensaio de Fluorescência por Polarização/métodos , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/virologia , Animais , Galinhas , Imunoensaio de Fluorescência por Polarização/instrumentação , Virus da Influenza A Subtipo H5N1/genética , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
Fluorescent polarization immunoassay (FPIA) is a single-step immunoassay method that is applicable to point-of-care testing; however, its applicability to large biomolecules has been restricted because ordinary FPIA is a competitive assay. Here, we report a noncompetitive FPIA using the variable domain from the heavy chain of a camelid antibody (VHH antibody). FPIA with VHH was successfully used to quantitate rabbit immunoglobulin G (IgG) and demonstrated a wider response range than that observed with antibody-binding (Fab) fragment. Then, using a portable FPIA instrument, a VHH-based immunoassay of human IgG in a human serum certified reference material was demonstrated.
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Imunoensaio de Fluorescência por Polarização/métodos , Imunoglobulina G/análise , Animais , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Limite de Detecção , Coelhos , Anticorpos de Domínio Único/imunologiaRESUMO
To examine the transport of an ionic substance through a bilayer lipid membrane (BLM), an electrochemical method combined with fluorometry was proposed. In this method, the transport of a fluorescent ion through the BLM was detected both as the transmembrane current and the dynamic change of fluorescence intensity synchronizing scanning membrane potential. The fluorescence intensity was measured in the local area close to the planar BLM by utilizing a confocal fluorescence microscope. The electrochemical method combined with fluorometry makes it possible to analyze only the transport of a target fluorescent ion in distinction from the transport of other coexisting ions. With the proposed electrochemical method, the ion transport caused by both a hydrophobic fluorescent cation (rhodamine 6G+, R6G+) and a relatively hydrophobic anion (BF4-) was examined. The electrochemical method combined with fluorometry characterized the transmembrane current as the transport of R6G+. Membrane conductance for the R6G+ transport increased proportionally to the concentrations of R6G+ and BF4- distributed in the hydrocarbon medium of the BLM which were estimated by extraction experiments with liposomes. These results show that the distribution of a cation and an anion from the aqueous phase in the BLM predominantly controls the membrane conductance for ion transport through the BLM.
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Compostos de Boro/química , Transporte de Íons , Bicamadas Lipídicas/química , Rodaminas/química , Colesterol/química , Técnicas Eletroquímicas/métodos , Fluorometria , Fosfatidilcolinas/químicaRESUMO
We investigated the sensor behavior of a field effect transistor, the channel of which is made of atomically thin MoS2 layers, focusing on the interaction of the MoS2 channel with the solution containing target molecules. For this purpose, we made a newly designed device in which the mask covered the electrodes of the source and the drain in order to make the solution contact only with the channel. In addition, a micro-fluid tank was fabricated above the channel as a solution reservoir. We examined the FET properties of this device for the sensing of the nicotine molecule for the development of a detection system for this molecule in the human body under in vivo conditions. We detected the sensor behavior both for the drop-cast process and for the condition where the channel contacts with the solution. The drain-current vs. gate-voltage variation of the MoS2-FET with the attachment of the nicotine molecule was clearly observed for both cases. For the latter case, the threshold voltage shifted in the negative gate-voltage direction with the increase of the concentration of the nicotine in the solution. This can be explained by the electron transfer from the molecule to the MoS2 channel, which was further confirmed by analyzing the X-ray photoemission spectroscopy and Raman spectroscopy together with the DFT calculation. The sensor can detect the variation of the nicotine concentration in the IPA solution by detecting the Vth change of the MoS2-FET.
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This work demonstrates that the solute concentration inside 100 micrometer-sized aqueous microdroplets can be controlled by adjusting the time required for the aqueous nanometer-sized droplets (nanodroplet) or reverse micelles to pass over the surface of the microdroplet. The kinetics of molecular transport between the microdroplets and the nanodroplets was investigated by utilizing a microdroplet array, and on the basis of these results, a control over the concentration selectivity of the contents of the microdroplet was achieved. This method is operationally simple and can be potentially applied as a pretreatment method for microanalytical systems that require high-density microdroplet arrays. This method can also be utilized for parallel small sample analyses such as single cell analysis.
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The distribution of ions into a bilayer lipid membrane (BLM) and their adsorption on the BLM are investigated by extracting a hydrophobic cation, rhodamine 6G (R6G+), into a liposome through the dialysis membrane method. R6G+ distribution mainly depends upon the concentration of the coexisting anion and its species (Cl-, Br-, BF4-, ClO4-, and picrate). On the other hand, R6G+ adsorption on the BLM surface follows the Langmuir adsorption model and is independent of the coexisting anion in the aqueous phase. We propose an extraction model of ionic species into the BLM, to explain the dependence of extraction of ionic species upon the coexisting anion. In this model, an ion is distributed with a coexisting counterion into the BLM and then forms an ion pair in the BLM. Here, the ion adsorption equilibrium on the BLM surface is independent of the species and concentration of the coexisting counterion under the same ionic strength. On the basis of this model, we estimate the distribution constant of R6G+ and anion (KD), the ion-pair formation constant in the BLM (Kip), and the R6G+ adsorption constant on the BLM surface (Kad). Even for an ultrathin membrane system, such as a BLM, R6G+ is distributed with a coexisting counterion and the distribution equilibrium of the ionic species at the water-BLM interface is analyzable similar to that at the water-organic solvent interface.
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The selective concentration of the contents in a microdroplet using spontaneous emulsification was proposed and demonstrated in a microfluidic channel. Aqueous microdroplets having a 40-µm diameter, in octane containing 100 mM of Span 80, shrank to 10 µm within 10 min with nanodroplet formation at the interface of the microdroplets. The microdroplets' contents either stayed in the microdroplet or partitioned into the nanodroplets, depending on their properties. The size and the hydrophobicity of the contents are two parameters that determine concentration/separation. In addition, this method was applied to a bound complex and free ligand (B/F) separation to demonstrate its applicability to biochemical analyses. Here we report the separation of water-soluble molecules in microdroplets for the first time. This method is expected to enhance the flexibility of the design of droplet analytical processes and widen their applicability.
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Emulsões/química , Corantes Fluorescentes/isolamento & purificação , Hexoses/química , Rodaminas/isolamento & purificação , Tensoativos/química , Água/química , Interações Hidrofóbicas e Hidrofílicas , Técnicas Analíticas Microfluídicas/métodosRESUMO
Spontaneous emulsification is a phenomenon that forms nanometer-sized droplets (nanodroplets) without the application of any external force, and the mechanism has been actively studied for application to various technologies. In this study, we analyzed the kinetics of spontaneous emulsification induced by Span 80. The measurement of water concentration in Span 80 hexadecane solution indicated that the chemical potential of water in the nanodroplets decreased as the amount of water in the nanodroplets decreased. Based on this result, water transport between the aqueous phase and nanodroplets in which the chemical potential of water was controlled was quantitatively investigated by using a microfluidic device. The results demonstrate that the kinetics of water transport during spontaneous emulsification induced by Span 80 was described by a model of osmotic transport through an organic liquid film between the aqueous phase and nanodroplets.
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Monitoring phycotoxin accumulation in marine products such as edible shellfish is a regulatory requirement in many countries. Therefore, a simple and rapid onsite quantification method is sought. Herein, we present a fluorescence polarization immunoassay (FPIA), a well-known one-step immunoassay, using a portable fluorescence polarization analyzer for domoic acid (DA), widely referred to as the primary toxin of amnesic shellfish poisoning (ASP). To establish FPIA for DA, the matrix effect of methanol, which is widely used to extract DA from shellfish, on FPIA was investigated. To validate this method, we performed a spike recovery test using oysters containing DA at a concentration equivalent to the regulatory limits of North America and the European Union (20 mg/kg). The recovery rate was found to be 79.4-114.7%, which is equivalent to that of the commercially available enzyme-linked immunosorbent assay (ELISA). We expect that this FPIA system will enable the quantitative onsite analysis of DA and significantly contribute to the safety of marine products.
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Toxinas Marinhas , Frutos do Mar , Toxinas Marinhas/análise , Imunoensaio de Fluorescência por Polarização , Frutos do Mar/análise , Alimentos Marinhos/análiseRESUMO
Microfluidic paper analytical devices (µPADs) are among the most promising platforms for heavy metal ion analysis. On the other hand, achieving simple and highly sensitive analysis of µPADs is challenging. In this study, we developed a simple enrichment method for sensitive multi-ion detection utilizing water-insoluble organic nanocrystals accumulated on µPAD. By combining the enrichment method with multivariate data analysis, three metal ion concentrations in the ion mixtures were simultaneously quantified with high sensitivity owing to the sensitive responses of the organic nanocrystals. In this work, we successfully quantified Zn2+, Cu2+, and Ni2+ at 20 ng L-1 in the mixed ion solution using only two dye indicators with a larger sensitivity improvement than those reported in previous studies. Interference studies revealed possibilities for a practical application in real sample analysis. This developed approach also can be used for other analytes.
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This paper is the first report of a non-competitive fluorescence polarization immunoassay (NC-FPIA) using a peptide as a tracer. The NC-FPIA can easily and quickly quantify the target after simply mixing them together. This feature is desirable for point-of-need applications such as clinical diagnostics, infectious disease screening, on-site analysis for food safety, etc. In this study, the NC-FPIA was applied to detect CD9, which is one of the exosome markers. We succeeded in detecting not only CD9 but also CD9 expressing exosomes derived from HeLa cells. This method can be applied to various targets if a tracer for the target can be prepared, and expectations are high for its future uses.
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Peptídeos , Polarização de Fluorescência , Imunoensaio de Fluorescência por Polarização/métodos , Células HeLa , Humanos , Tetraspanina 29RESUMO
Aerosol droplets play a critical role in the development of weather patterns, yet are notoriously difficult to analyze because of their small size, transient nature and potentially complex composition. As a result, there has been a surge in recent years in the development of analysis techniques aimed at the study of aerosol droplets, namely of their surface tension properties, which are thought to play a great role in aerosol/cloud growth and subsequently having an impact on the resulting weather patterns. To capture the state of the field at this key time, we have collected and described some of the most relevant and influential studies, with a focus on those that have had the most impact. This review will present and describe the most used analytical techniques for studying the surface tension of micrometer-sized aqueous droplets, with a focus on historical trends and how the current techniques are posed to revolutionize the field.
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The partitioning of water and tetramethylrhodamine-conjugated-10-residue oligopeptides from the aqueous phase of microdroplets into Span 80 reverse micelles was observed by utilizing microdroplet arrays. Each peptide was dissolved in phosphate buffer saline, and initially encapsulated in arrayed droplets. An organic phase containing the reverse micelles was added to the microdroplets. Here, the hydration degree of the reverse micelle was adjusted by contact of the organic phase with a 1.0 M NaCl aqueous solution or with a phosphate buffer saline before combining it with the microdroplets. For micelles treated with a 1.0 M NaCl, significant water transport from the microdroplet to the micelle was observed, and peptide with low solubility in water was transported to the reverse micelles, while those with high solubility in water were not. For micelles treated with phosphate buffer saline, the water transport was minimal, and no significant peptide transport was observed. These results suggest that the partitioning of low-solubility oligopeptides requires accompanying water transport to the reverse micelle phase.
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Micelas , Tensoativos , Hexoses , Oligopeptídeos , ÁguaRESUMO
The absorption spectra of bromothymol blue (BTB) solution introduced in microfluidic devices were reproduced by principal component analysis (PCA)-based xyz-spectrum conversion methods for geometric mapping of the pH values of fluids. We fabricated PDMS-made microfluidic devices with a channel depth of 1 mm to overcome the lower detection limits of transmittance image acquisition. Aqueous solutions of pH indicators under various pH conditions were hydrodynamically introduced into the channel, and RGB values of the region of interest (ROI) were obtained via image analysis. The xyz values were then converted into absorption spectral data of the pH indicator using the PCA-based spectral reproduction previously proposed by the authors. The high reproducibility of the spectra was confirmed to be comparable to that of the conventional method using a spectrophotometer. We applied the present method to elucidate the pH gradient at an aqueous biphasic interface in the microfluidic channels generated by contacting multiple laminar flows of two or three buffered solutions. We confirmed that the pH gradient ranged from approximately 70 to 140 µm, which is consistent with the results reported using other approaches. The results demonstrate the applicability of the present method to the fluctuation field in micro/nanospaces to acquire spectrophotometric information in the order of milliseconds without monochromating equipment.