Detection of Fibril Nucleation in Micrometer-Sized Protein Condensates and Suppression of Sup35NM Fibril Nucleation by Liquid-Liquid Phase Separation.

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.

Nanoparticle Assembly at the Water-Oil Interface Induced by Spontaneous Emulsification for Microdroplet Immunoassay.

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.

A microfluidic approach for the detection of uric acid through electrical measurement using an atomically thin MoS2 field-effect transistor.

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.

In Situ Surface Tension Measurement of Deliquesced Aerosol Particles.

The surface tension of aerosol particles can potentially affect cloud droplet activation. Hence, direct measurement of the surface tensions of deliquesced aerosol particles is essential but is challenging. Here, we report in situ surface tension measurements based on a novel method that couples a linear quadrupole electrodynamic balance (EDB) with quasi-elastic light scattering (QELS). The EDB-QELS is validated using surface tension measurements of atmospherically relevant inorganic and organic droplets. The surface tension results reasonably agree with the reference values in the range of ∼50-90 mN m-1. We find a significant size dependence for sodium chloride droplets containing surface-active species (sodium dodecyl sulfate) in the size range of ∼5-18 µm. The surface tension increases from ∼55 to 80 mN m-1 with decreased size. Relative humidity (RH)-dependent surface tensions of mixed ammonium sulfate (AS) and polyethylene glycol droplets reveal the onset of liquid-liquid phase separation. Droplets containing water-soluble matter extracted from ambient aerosol samples and 2.3-2.9 M AS exhibit a ∼30% reduction in surface tension in the presence of ∼50 mmol-C L-1 water-soluble organic carbon, compared to pure water (∼72 mN m-1). The approach can offer size-resolved and RH-dependent surface tension measurements of deliquesced aerosol particles.

Non-competitive fluorescence polarization immunoassay for detection of H5 avian influenza virus using a portable analyzer.

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.

Noncompetitive Fluorescence Polarization Immunoassay for Protein Determination.

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.

Microfluidic tank assisted nicotine sensing property of field effect transistor composed of an atomically thin MoS2 channel.

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.

Rapid detection of anti-H5 avian influenza virus antibody by fluorescence polarization immunoassay using a portable fluorescence polarization analyzer.

A rapid, facile and selective detection of anti-H5 subtype avian influenza virus (AIV) antibody in serum by fluorescence polarization immunoassay (FPIA) was achieved. A fragment of recombinant H5 subtype AIV hemagglutinin was produced and labeled with fluorescein to use it as a labeled antigen in FPIA. This labeled antigen was mixed with anti-AIV sera (H1-H16 subtypes) and FP of the mixture was measured using a portable FP analyzer on a microdevice. It was found that FP increased in proportion to the concentration of anti-H5 AIV antibody (serum) and was significantly higher than FP obtained with the other sera. The selective detection of anti-H5 subtype AIV antibody was confirmed. The required volume of original sample was 2 µL and analysis time was within 20 min. This detection system realizes an efficient on-site diagnosis and surveillance of AIV.

Kinetic Switching of the Concentration/Separation Behavior of Microdroplets.

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.

Optical Surface Tension Measurement of Two-Dimensionally Confined Liquid Surfaces.

Surface tension measurement based on spontaneous capillary wave resonance in confined micrometer-sized liquid interfaces was demonstrated. A single-beam quasi-elastic laser scattering method was used to detect the resonance. Characteristic resonant modes were observed on a 44-µm-sized circular water surface. The frequencies of the peaks agreed well with those simulated by assuming planar resonance, and the relationship was further confirmed for triangular, square, and pentagonal water surfaces. Then, the applicability of the method was successfully demonstrated by surface tension measurements of aqueous solutions of sodium dodecyl sulfate. The sensitive detection of capillary resonance opens new possibilities for the chemical and biochemical analysis of liquid interfaces.

Microfluidic selective concentration of microdroplet contents by spontaneous emulsification.

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.

Fluorescence polarization measurement system using a liquid crystal layer and an image sensor.

The detection system which enables simultaneous fluorescence polarization (FP) measurement of multiple samples was proposed and proven by a proof-of-concept experiment on the viscosity dependence of FP of fluorescein sample in water-ethylene glycol solution and another experiment on the FP immunoassay of prostaglandin E2 sample. The measurement principle of FP is based on the synchronization between the orientation of the liquid crystal molecules and the sampling frequency of a CCD. This report is the first description of the simultaneous FP measurement of multiple samples. This system has a great potential for equipment miniaturization and price reduction as well as providing simultaneous FP measurement of multiple samples.

Kinetic description of water transport during spontaneous emulsification induced by Span 80.

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.

Rapid determination of domoic acid in seafood by fluorescence polarization immunoassay using a portable analyzer.

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.

Sensitive and simple multi-ion detection using organic nanocrystal enrichment in paper analytical devices.

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.

Surface tension measurement at the microscale by passive resonance of capillary waves.

The properties of fluid interfaces increase in importance as the physical scale decreases and, hence, characterization of surface tension becomes all the more critical. However, there is to date no method to characterize this parameter on microscale surfaces. We propose here a simple method based on the resonance of capillary waves, which are naturally excited by thermal fluctuations, under one-dimensional spatial restrictions using single-beam dynamic light scattering. The principle was verified at methanol/air interfaces in polydimethylsiloxane (PDMS) microchannels having various widths. Characteristic comb-shape power spectra were experimentally obtained. Theoretical analysis showed that the spectral peaks correspond to the first or higher modes of the capillary wave resonance in the restricted space between the parallel channel walls. A useful relation between successive modes was derived to eliminate the effects of damping at the soft PDMS walls. Thus, for methanol, two values were calculated from three successive modes (24.8 and 21.2 mN/m); the literature value is 22.02 mN/m. For acetonitrile, the value obtained was 28.2 ± 5 mN/m, close to the literature value of 28.6 mN/m. Although accuracy and precision require further elucidation, this novel method is expected to become a powerful tool at the micro/nanoscale.

Non-competitive fluorescence polarization immunosensing for CD9 detection using a peptide as a tracer.

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.

Transport of Oligopeptide from Aqueous Phase to Span 80 Reverse Micelles in Microdroplet Array.

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.

Aerosol Droplet Surface Measurement Methods.

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.

Controlling water transport between micelles and aqueous microdroplets during sample enrichment.

Droplet microfluidics technologies have advanced rapidly, but enrichment in droplets has still been difficult. To deterministically control the droplet enrichment, the water transport from an aqueous microdroplet in organic continuous phase containing span 80 micelles was investigated. Organic phase containing Span-80-micelles contacted a NaCl aqueous solution to control hydration degree of the micelles, prior to being used in the microfluidic device. Then, the organic phase was continuously applied to the microdroplets trappled in microwells. Here, water was transported from the microdroplet to the organic phase micelles. This spontaneous emulsification process induced the droplet shrinkage and stopped when the microdroplet reached a certain diameter. The micelle hydration degree correlated well with the final water activity of droplets. The enrichment factor can be determined by the initial microdroplet salt concentration and by the micelle hydration degree. As a proof-of-concept experiment, enrichment of fluorescent nanoparticles and dye was demonstrated, and fluorescent resonance energy transfer was observed as expected. Another demonstration of bound-free separation was performed utilizing the avidin-biotin system. This technique has the potential to be a powerful pretreatment method for bioassays in droplet microfluidics.