Probing the interaction between biomolecules under sub-zero temperature conditions by electrophoresis in ice grain boundaries.

BACKGROUND: Psychrophiles can survive under cryogenic conditions because of various biomolecules. These molecules interact with cells, ice crystals, and lipid bilayers to enhance their functionality. Previous studies typically measured these interactions by thawing frozen samples and conducting biological assays at room temperature; however, studying these interactions under cryogenic conditions is crucial. This is because these biomolecules can function at lower temperatures. Therefore, a platform for measuring chemical interactions under sub-zero temperature conditions must be established. RESULTS: The chemical interactions between biomolecules under sub-zero temperature conditions were evaluated within ice grain boundaries with a channel-like structure, which circumvents the need for thawing. An aqueous solution of sucrose was frozen within a microfluidic channel, facilitating the formation of freeze-concentrated solutions (FCSs) that functioned as size-tunable electrophoretic fields. Avidin proteins or single-stranded DNA (ssDNA) were introduced into the FCS in advance. Probe micro/nanospheres whose surfaces were modified with molecules complementary to the target analytes were introduced into the FCS. If the targets have functionalities under sub-zero temperature conditions, they interact with complementary molecules. The chemical interactions between the target molecules and nanospheres led to the aggregation of the particles. The size tunability of the diameter of the FCS channels enabled the recognition of aggregation levels, which is indicative of interaction reactivity. The avidin-biotin interaction and ssDNA hybridization served as models for chemical interactions, demonstrating interactivity under sub-zero temperature conditions. The results presented herein suggest the potential for in situ measurement of biochemical assays in the frozen state, elucidating the functionality of bio-related macromolecules at or slightly below 0 °C. SIGNIFICANCE: This is the first methodology to evaluate chemical interactions under sub-zero temperature conditions without employing the freeze-and-thaw process. This method has the advantage of revealing the chemical interactions only at low temperatures. Therefore, it can be used to screen and evaluate the functionality of cryo-related biomolecules, including cold-shock and antifreeze proteins.

Elucidating the Quenching Mechanism of Tris(2,2'-bipyridyl)ruthenium(II) Complex in the Water-Glycerol Binary System Based on the Microscopic Structure of the Media.

Kinetic studies on the photochemical quenching reaction of the tris(2,2'-bipyridyl) ruthenium(II) complex ([Ru(bpy)3]2+) in water-glycerol binary media were conducted based on the Einstein-Smoluchowski (E-S) theory. Dynamic and static quenching behaviors were analyzed by comparing results from time-resolved spectroscopy and emission spectroscopy. While the dynamic quenching reaction aligns well with the E-S theory, static quenching was observed, leading to a notable increase in the overall photoquenching reaction rate constant. Employing chromatography and infrared spectroscopy, we correlated the microscopic molecular structure of the binary solvent system and the solvation environment around the emitters with the reaction mechanism. This correlation was found to correspond to ion pair formation and the confinement effect of the emitter, respectively.

Absorption-based colorimetric detection of nickel(II) ion by phase separation of thermoresponsive magnetic nanoparticles under microflow.

Therma-Max™ LSA Streptavidin is a thermoresponsive magnetic nanoparticle (TMNP). It can be introduced conveniently to molecular recognition groups by avidin-biotin interaction. In this study, we demonstrated the detection of nickel(II) ions by the magnetic separation of TMNP induced by their phase transition under microflow. The NTA-tagged TMNP solution mixed with a Ni2+ sample was introduced into a microchannel with a well structure. Moreover, the sample was heated to induce the thermally induced aggregation of TMNP. The Ni-capturing TMNP were trapped in the well by magnetic fields. The supernatant was removed from the outlet, and a dimethylglyoxime (DMG) solution was introduced into the device for colorimetric detection in the well. Because DMG has a higher stability constant with Ni2+, sensitive colorimetric detection of Ni2+ can be achieved in devices where the sample volume, e.g., optical pathlength, is short. To demonstrate the feasibility of the proposed method, a recovery test was conducted using a commercially available cosmetic sample. Therein, complete collection was achieved.

Element-ratio dependence of the 5d-states of Au and Pt in solid-solution-type Au-Pt alloy nanoparticles studied by X-ray absorption spectroscopy and density functional theory.

Solid-solution-type Au-Pt alloy nanoparticles (NPs) were prepared from the nanoclusters of each metal using the polymer-conjugated fusion growth method. The elemental mapping analysis showed that the mixing state of the elements in the NPs drastically changed in the narrow reaction-temperature range from 100 °C to 180 °C. For their various mixing states, the 5d-states of Au and Pt atoms in the alloy NPs were investigated on the basis of the white line intensities of X-ray absorption near edge structure (XANES). Then, the 5d-states of Au and Pt atoms in a model crystalline ordered alloy structures were investigated on the basis of the theoretically calculated XANES spectra using density functional theory (DFT) in the whole composition range. The DFT calculation showed that the changes in the absorption spectra near the Pt and Au edges are caused by the change in the occupation of the Pt 5d-states and the orbital hybridisation of the Au 5d-states with the 5d-states of neighbouring Pt atoms around an Au atom, respectively.

Incorporation of a Morphologically Controlled Ice Grain Boundary into a Microfluidic Device for Size-Selective Separation of Micro/Nanospheres.

A frozen aqueous solution was integrated into a microfluidic device as a size-tunable separation field for the size-selective separation of micro/nanospheres. The width of the ice grain boundaries formed in frozen aqueous solutions could be altered by controlling the operating temperature. A freezing chamber was placed adjacent to the microfluidic channel. A sample-dispersing aqueous sucrose solution was injected into the chamber and frozen, allowing the freeze-concentrated solution (FCS) to run vertically to the microfluidic channel, where the eluting solution flows. The operating temperature can be used to control the physical interaction between the ice wall and micro/nanospheres, enabling size-selective migration. The eluted micro/nanospheres in the microchannel were passed through the eluting solution collected from the outlet. We achieved size-selective separation and collection of microspheres and nanospheres. We separated the exosomes and yeast cells to demonstrate their applicability in bioseparation. The present method is suitable not only for size-selective separation but also for evaluating the biological expression of extracellular vesicles under cryogenic conditions.

Rapid acquisition of absorption spectra to monitor proton migration in nanoliter space using the RGB-spectrum-conversion method with a 10 ms interval.

Acquisition protocol of absorption spectra at nanoliter spaces from the RGB values preserved in video data at 10 ms intervals was established using the principal-component-analysis-based RGB-conversion method. Proton behavior was monitored using a camera to acquire the video footage to monitor colorimetric change in the nanoliter space. The RGB values observed in the video were converted into a score vector using a conversion matrix. A linear combination of the predetermined loading vectors with the score values was calculated to reproduce the absorption spectra. The reproduced absorption spectra correlated well with those acquired using a conventional spectrophotometer during a short period. This method was applied to monitor the proton diffusion from a single cationic ion-exchange resin into hydrogels at low concentrations. The rapid acquisition and quick response of this method may enable the monitoring of the initial diffusion of protons, which is challenging with conventional spectrophotometry and electrochemical approaches.

Fabrication of paper-based analytical devices by a laminating method with thermal ink ribbons, sticky notes, and office appliances.

A stencil printing method utilizing sticky notes, a thermal transfer ink ribbon, and office appliances for paper-based analytical device (PAD) fabrication was proposed. A sticky note was attached to a filter paper, and a mask pattern was cut using a cutting machine. A commercially available thermal ink ribbon was then placed over the mask and laminated. We have characterized the fabricated devices. This approach could be used for the fast and mass prototyping of PADs using simple office appliances with no need for a wax printer.

Charge-Selective Aggregation Behavior of Thermoresponsive Polyelectrolytes Having Low Charge Density in Aqueous Solutions of Organic Counterions.

The aggregation behavior of thermoresponsive polyelectrolytes with low charge density in aqueous solutions of organic counterions was investigated. We synthesized two thermoresponsive polyelectrolytes: anionic poly(N-isopropylacrylamide-co-(3-sulfopropyl)acrylamide potassium) (P-NIP-SPAK) and cationic poly(N-isopropylacrylamide-co-(3-acrylamidepropyl)trimethylammonium chloride) (P-NIP-AAPTAC). The polyelectrolytes remained soluble in their aqueous solutions even above the lower critical soluble temperature of P-NIP owing to the strong hydration property of the ionic groups. The aggregation occurred when organic counterions were added to the solution. In these solution systems, the concentration of counterions exceeds those of ionic groups introduced into the polyelectrolytes. The aggregation behavior is attributed to the salting-out effect of counterions accommodated near the polyelectrolyte surface by electrostatic interaction. This aggregation behavior was utilized for the charge-selective recognition of amino acids. P-NIP-SPAK aggregated only when basic amino acids were added under acidic conditions, whereas P-NIP-AAPTAC aggregated only when acidic amino acids were added under basic conditions. The results herein demonstrate that P-NIP-SPAK and P-NIP-AAPTAC have the potential to be used as charge-selective polymer sensors for amino acids without having to strictly control the experimental conditions.

Total protein assay by PCA-based RGB-spectrum conversion methods with smartphone-acquired digital images.

Total protein concentrations in the aqueous solutions were determined from the absorption spectra reproduced from smartphone-captured digital color images. We employed two different procedures for protein determination: the pyrogallol red molybdate method and Bradford's method. The principal-component-analysis-based reproduction process, which was previously reported by our research group, enabled the conversion of RGB values to score values for a linear combination of loading vectors to generate reproduced absorption spectra. The reproduced spectra were identical to those measured using a commercially available spectrophotometer. The total protein assays of commercial soymilk and human serum samples were carried out with both coloration reagents, and the obtained results were in good agreement with those attained using a conventional spectrophotometer. These results show that the proposed method enables smartphone-based ratiometric analysis of real samples without requiring any monochromating equipment.

Geometrical pH mapping of Microfluids by principal-component-analysis-based xyz-spectrum conversion method.

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.

Dataset for reproducing absorption spectra of methyl orange from the RGB values of microscopic images.

The present dataset is related to the research paper entitled "Reproducing Absorption Spectra of pH Indicators from RGB Values of Microscopic Images" (Inagawa et al., 2020). The dataset contains microscopic images of aqueous methyl orange (MO), absorption spectra acquired with a spectrophotometer, loading spectra and calculation sheets for reproducing absorption spectra of aqueous MO from their RGB values of the microscopic images. The microscopic transmission images of the standard MO solutions at various pH conditions were acquired with a CMOS camera equipped with an invert microscope. Meanwhile, the loading spectra were obtained by principle component analysis of a series of absorption spectra of the standard solutions. The conversion matrix from RGB values in a region of interests (ROI) to score values were linear-algebraically determined from the RGB values and score values of the standard solutions. The absorption spectra of the sample solutions of which pH conditions are unknown were then reproduced by calculating the linear combination of the loading spectra with the score values obtained from the conversion process. Herein, the absorption spectra of MO are reproduced at various pH and ROI conditions.

Reproducing absorption spectra of pH indicators from RGB values of microscopic images.

Absorption spectra of pH indicators in aqueous solutions were reproduced from RGB values of microscopic images utilizing principal component analysis (PCA) and linear algebraic treatments. The reproduction of absorption spectra comprises the following three steps: (1) determining the loading spectra by PCA, (2) determining the conversion matrix from the RGB values to the score vectors, and (3) reproducing the absorption spectra by linear combination of the loading spectra and the score vectors. The reproducibility of the absorption spectra was demonstrated by employing bromothymol blue and methyl red solutions as pH indicators. The reproduced spectra of both indicators were in good agreement with the spectra measured with a conventional spectrophotometer. The pKa values of both indicators calculated from the reproduced spectra are in good agreement with those obtained from the spectrophotometric spectra and the literature values, confirming validity of the reproduction. This approach was applied to measure pH of freeze concentrated solutions in micro drains formed in ice. A change in pH was successfully observed on freezing and was compared with that reported in previous literature. Since this method does not necessitate the use of grating systems, spectral changes can be traced in milliseconds; this elucidates the phenomena occurring in fluctuating fields.

Interaction between antifreeze protein and ice crystal facet evaluated by ice-channel electrophoretic measurements of threshold electric field strength.

The chemical interaction between antifreeze proteins (AFPs) and ice crystals is evaluated via electrophoresis of AFP-anchored microparticles in fluidic channels formed in frozen aqueous sucrose. Straight fluidic channels are created in a flat glass chamber connecting two Ag/AgCl electrodes. This configuration allows us to estimate an electric field strength exerted on probe particles migrating along the channel. When the channel width is comparable to the particle size, the particle is immobile because of the resistance force induced by the interaction with the ice wall. However, when the overall electrophoretic force surpasses the resistance force, the microsphere starts to migrate. From the threshold electric field strengths determined for unmodified and AFP-modified particles, the resistance forces for the chemical interaction between AFPs and ice wall are estimated.

Interaction potential surface between Raman scattering enhancing nanoparticles conjugated with a functional copolymer.

A novel Raman scattering enhancement was discovered using colloid nanoparticles conjugated with an amine-based copolymer. The interaction potential surface between Raman scattering enhancing nanoparticles was clarified by combining a small-angle scattering method and a model-potential-free liquid-state theory as an in situ observation in the solution state. The potential surface indicates that the most stable position is located around 0.9 nm from the particle surface, suggesting the existence of a nanogap structure between the nanocomposites. The change in Raman scattering enhancement was also acquired during the dispersion process of the aggregated nanocomposites through a glutathione-triggered nanosensing reaction.

Thermal-induced Immuno-nephelometry Using Gold Nanoparticles Conjugated with a Thermoresponsive Polymer for the Detection of Avidin.

Thermoresponsive immunonephelometry was achieved with biotinylated poly(acrylate) and thermoresponsive gold nanocomposites composed of 13-nm gold nanoparticles and thermoresponsive polymers containing triethylenetetramine and biotin groups. The avidin-biotin interaction was used to model an immunoreaction in order to demonstrate thermoresponsive immunonephelometry. In the absence of avidin, positively charged gold nanocomposites electrostatically interacted with biotinylated poly(acrylate) to form binary complexes, in which the charges canceled each other out. The charge cancelation resulted in the binary complexes precipitating when the solution was heated above the phase-transition temperature. However, adding avidin formed ternary sandwich complexes through the avidin-biotin interaction. The ternary complexes remained sufficiently soluble above the phase-transition temperature because of the spatial isolation of the positive and negative charges. The transmittance of the solution containing the thermoresponsive gold nanocomposites and biotinylated poly(acrylate) at 37°C increased as the avidin concentration increased. A sigmoidal profile was observed from 10(-6.5) to 10(-5.5) mol/L. The concentration of avidin spiked in bovine serum was determined by our method.

Interaction of poly(N-isopropylacrylamide) with sodium dodecyl sulfate below the critical aggregation concentration.

Interaction between the thermoresponsive polymer poly(N-isopropylacrylamide) (P-NIP) and sodium dodecyl sulfate (SDS) both above and below its phase transition temperature was examined under dilute conditions. Above the lower critical solution temperature (LCST) of P-NIP (32 °C), 0.01 wt % P-NIP specifically interacted with 1.0 × 10(-5) mol/L SDS to form a precipitate. However, when SDS was added at concentrations above or below 1.0 × 10(-5) mol/L, the P-NIP solution remained clear above the LCST. A fluorometric probe, N-phenyl-naphthalene, indicated that the hydrophobicity of the aggregates composed of P-NIP and SDS changed at an SDS concentration of 1.0 × 10(-5) mol/L. Although the hydrophobicity of the precipitate was similar to that of P-NIP alone at less than 1.0 × 10(-5) mol/L, it approached that of SDS homomicelles as the SDS concentration increased above 1.0 × 10(-5) mol/L. Dynamic light scattering and turbidimetry studies showed no P-NIP phase transition above an SDS concentration of 1.0 × 10(-5) mol/L, which is much lower than the reported critical association concentration (CAC) of SDS with P-NIP. This indicates that P-NIP interacted with SDS above the LSCT at much lower SDS concentration than the reported CAC.

Release of Nile red from thermoresponsive gold nanocomposites by heating a solution and the addition of glutathione.

Thermoresponsive gold nanocomposites encapsulating Nile red were fabricated by the conjugation of gold nanoparticles containing Nile red with a thermoresponsive polymer, poly(N-isopropylacrylamide(90 mol%)-co-N-acryloyldiethylenetriamine(10 mol%)). They were then examined as a model of drug delivery carriers and colloidal fluorescence sensors. Nile red, as a fluorophore to be released, was introduced to the surface of gold nanoparticles prior to conjugation with thermoresponsive polymers. Heating a solution at 90°C resulted in shrinkage of the thermoresponsive polymers, which facilitated disassembly of the gold nanocomposites in the presence of glutathione. This disassembly caused a replacement of Nile red with glutathione at the surface of the gold nanoparticles, followed by the release of Nile red from the gold nanocomposites. Nile red liberated from the gold surface recovered its inherent fluorescence properties that had been quenched by gold nanoparticles through fluorescence resonance energy transfer. The fluorescence intensity of the liberated Nile red increased linearly as the glutathione concentration increased up to 1.0 × 10(-5) mol/L, demonstrating that thermoresponsive gold nanocomposites can be used as colloidal sensors or drug delivery carriers that can be manipulated by the concentration of glutathione and the solution temperature. The applicability of the thermoresponsive gold nanocomposites to colloidal fluorescence probes was also checked by assay of glutathione in tablets.

Thermal-induced growth of gold nanoparticles conjugated with thermoresponsive polymer without chemical reduction.

The growth of gold nanoparticles without chemical reduction of gold (III) ions was achieved by the disruption of thermoresponsive polymers conjugated with the gold nanoparticles through the phase transition of the polymers. When a solution of gold nanoparticles coated with thermoresponsive polymers was heated, chains of the thermoresponsive polymers were disrupted because of dehydration, resulting in the fusion of gold nanoparticles to form larger nanoparticles. The evolution of the extinction band around 550 nm evidenced the formation of these large (post-fusion) gold nanoparticles, which were characterized by transmission electron microscope (TEM) and dynamic light scattering (DLS). TEM images verified the formation of the large gold nanoparticles having particle sizes of 80-100 nm, whereas DLS indicated the existence of large nanoparticles with hydrodynamic diameters exceeding 200 nm. The deposition did not require the addition of reductants or trivalent gold ions for the formation of the large gold nanoparticles. Both the heating and the solution conditions were studied to elucidate the mechanism of the formation of large gold nanoparticles.

Polymer-functionalized gold nanoparticles as versatile sensing materials.

In this brief review, gold nanoparticles conjugated with functional polymers are described from the viewpoint of application to sensing materials. The optical properties of gold nanoparticles, the synthesis of polymer-functionalized gold nanoparticles, and their analytical applications are discussed. Polymer-functionalized gold nanoparticles are categorized into two classes: biopolymer-conjugated gold nanoparticles and artificial-polymer conjugated gold nanoparticles. Fluorometric and colorimetric sensing using gold nanoparticles are focused; fluorometric detection enables us to exploit sensitive assays for practical use. Furthermore, chemical amplification using gold nanoparticles is also discussed for the sensitive probing.

Colorimetric assay of glutathione based on the spontaneous disassembly of aggregated gold nanocomposites conjugated with water-soluble polymer.

This article describes the glutathione-triggered disassembly of gold nanocomposites composed of gold cores and water-soluble copolymers [poly(N-n-isopropylacrylamide-co-acryloyldiethyletriamine)] attached to the surfaces of gold cores. The gold nanocomposites exhibit a bluish purple color because of the assembled gold cores that are conjugated with the diethylenetriamine groups incorporated into the copolymers. Glutathione added to the gold nanocomposite solution adsorbs onto the surface of the gold cores to liberate diethylenetriamine groups, resulting in spontaneous disassembly that changes the color of the solution to a reddish shade. Increasing the glutathione concentration facilitates the spontaneous disassembly of the gold nanocomposites. For the determination of glutathione, the colorimetric change of the gold nanoparticles is quantified with the a* value of the L*a*b* color coordinates defined by the CIE (Commission Internationale de l'Eclairage) chromaticity diagram. A linear relationship between the a* value and the glutathione concentration of up to 6 x 10(-6) mol/L is obtained 15 min after the addition of glutathione that has a detection limit (defined as 3sigma) of 2.9 x 10(-8) mol/L. The colorimetric assay is successfully applied to the determination of glutathione in eye drops and health supplements.