Diffusion behavior of rhodamine 6G in single octadecylsilyl-functionalized silica particle revealed by fluorescence correlation spectroscopy.

We investigated the diffusion behavior of rhodamine 6G (Rh6G) within single octadecylsilyl-functionalized (ODS) silica particle in an acetonitrile (ACN)/water system using fluorescence correlation spectroscopy (FCS). FCS measurements were conducted at the center of the particle to exclusively determine the intraparticle diffusion coefficient (D). The obtained D values were analyzed based on a pore and surface diffusion model, the results of which indicate that surface diffusion primarily governs the intraparticle diffusion of Rh6G. Furthermore, an increase in the concentration of ACN (CACN) resulted in a corresponding increase in the surface diffusion coefficient (Ds), whereas the addition of NaCl did not significantly affect the Ds values. We attributed this dependence of Ds to the dielectric constant change in the interfacial liquid phase formed on the ODS layer. Specially, Ds values of (4.0 ± 0.5) × 10-7, (7.7 ± 1.1) × 10-7, (1.0 ± 0.3) × 10-6, and (1.1 ± 0.2) × 10-6 cm2 s-1 were obtained for CACN = 20, 30, 40, and 50 vol%, respectively. We anticipate that this approach will contribute to advancing research on intraparticle mass transfer mechanisms.

Evolution of myoglobin diffusion mechanisms: exploring pore and surface diffusion in a single silica particle.

This study elucidates the mass transfer mechanism of myoglobin (Mb) within a single silica particle with a 50 nm pore size at various pH levels (6.0, 6.5, 6.8, and 7.0). Investigation of Mb distribution ratio (R) and distribution kinetics was conducted using absorption microspectroscopy. The highest R was observed at pH 6.8, near the isoelectric point of Mb, as the electrostatic repulsion between Mb molecules on the silica surface decreased. The time-course absorbance of Mb in the silica particle was rigorously analyzed based on a first-order reaction, yielding the intraparticle diffusion coefficient of Mb (Dp). Dp-(1 + R)-1 plots at different pH values were evaluated using the pore and surface diffusion model. Consequently, we found that at pH 6.0, Mb diffused in the silica particle exclusively through surface diffusion, whereas pore diffusion made a more substantial contribution at higher pH. Furthermore, we demonstrated that Mb diffusion was hindered by slow desorption, associated with the electrostatic charge of Mb. This comprehensive analysis provides insights into the diffusion mechanisms of Mb at acidic, neutral, and basic pH conditions.

Dynamic Interfacial Tension Measurement for the Kinetic Study of Eu(III) Extraction in the NaNO3/Tributyl Phosphate System Based on an Increase in Interfacial Tension.

We demonstrated for the first time that interfacial tension measurements can be used to evaluate the kinetics of the solvent extraction of metal ions. The Eu(III) extraction mechanism in the nitrate ion/tributyl phosphate (TBP) system was investigated on the basis of dynamic interfacial tension. Interestingly, the interfacial tension of the TBP droplet (γ) increased with Eu(III) extraction. This behavior can be explained by the electrocapillary effect. The time dependence of γ was kinetically analyzed, and we demonstrated that the rate-determining process was the interfacial reaction of Eu(III). Furthermore, the dependence of the mass transfer rate constant upon the concentration of the nitrate ions revealed that two nitrate ions were involved in the interfacial reaction during Eu(III) extraction. However, no change in the rate constant upon TBP concentration was observed, because the change in the TBP concentration did not affect the electrocapillary effect. We determined the forward and backward reaction rates to be k1 = (1.5 ± 0.7) × 10-6 m M-2 s-1 and k-MT = (6.9 ± 3.9) × 10-7 m s-1. Therefore, for the first time, we demonstrated that dynamic interfacial tension, which is involved in the electrocapillary effect, can be used to elucidate the kinetics of Eu(III) extraction. We expect that this study will attract the attention of researchers in several fields, including physical and analytical chemistry.

Kinetic detection of hydrogen peroxide in single horseradish peroxidase-concentrated silica particle using confocal fluorescence microspectroscopic measurement.

In the present study, we propose a scheme for detecting H2O2 by using horseradish peroxidase (HRP) adsorbed onto single silica particles and fluorescence microspectroscopy. When the silica particles were immersed in an HRP solution, the HRP concentration in the silica particles increased by a factor of 690 compared to that in the bulk aqueous solution because HRP was adsorbed on the silica surface. When a single particle containing HRP was added to a mixed solution of H2O2 and Amplex Red, fluorescence from resorufin, which was produced by the reaction of HRP, H2O2, and Amplex Red, was observed. The fluorescence from the resorufin in the particles increased after a single particle was added to the solution, and the release of resorufin was observed. As the concentration of H2O2 (CH2O2) decreased, the time it takes for fluorescence intensity to reach its maximum was shorter. The detection limit for H2O2 in the present system was 980 nM. The reaction behavior of a single silica particle was evaluated using a spherical diffusion model, which explains the approximate concentration change of resorufin in the silica particle. The proposed method has the advantages of simple sample preparation and detection, low sample consumption, and a short detection time.

Molecular crowding effect in Hantzch pyridine synthesis in polyethylene glycol aqueous solution.

In a molecular crowding environment, the kinetics and thermodynamics differ from those in a diluted solution. Although the molecular crowding effect has been extensively investigated, its fundamental kinetics and thermodynamics remain unclear. In this study, we investigated the change in the rate constant (k) of the Hantzch pyridine reaction in a molecular crowding environment using polyethylene glycol (PEG). While the k value increased to a PEG concentration (CPEG) of 10 vol%, a decreasing trend was observed for CPEG > 20 vol%. This intriguing behavior was analyzed based on the increase in reactant activity due to volume exclusion and the decrease in water activity due to osmotic pressure. Volume exclusion and osmotic pressure had opposing effects on the reaction, which were positive for volume exclusion and negative for osmotic pressure. We found that k decreased when the negative effect of the osmotic pressure surpassed the volume exclusion effect.

Investigating hydrophobic environment in alkyl-group-functionalized silica particle with various chain lengths using absorption microspectroscopy.

A well-known solvatochromic dye, Reichardt's dye (R-dye), was used to evaluate the hydrophobicity of alkyl-group-functionalized silica particles (ASPs) with different chain lengths. The absorption spectra of R-dye were measured in a single ASP in a mixed solution of water and an organic solvent (methanol (MeOH), ethanol (EtOH), acetonitrile (ACN), tetrahydrofuran (THF), or N,N-dimethylformamide (DMF)) using absorption microspectroscopy. The polarity parameter in the ASPs (ET), determined by the absorption maximum, was observed to be smaller than those in bulk solutions, indicating that R-dye was present in a more hydrophobic environment. In EtOH, THF, and DMF, R-dye was distributed within the alkyl chain layer including the organic solvent. An increase in the organic solvent content of the bulk solution led to a higher organic solvent concentration in the alkyl chain layer, resulting in a decrease in ET. In MeOH and ACN, the R-dye was distributed within the alkyl chain layer and concentrated phase. Moreover, with the increase in the organic molecule content, the distribution of R-dye in the concentrated phase became dominant in MeOH and ACN system, leading to an increase in the ET value. The findings presented in this paper are expected to attract the attention of a wide range of researchers in chromatography.

Kinetic analysis of mass transfer of Eu(III) in extractant-impregnated polymer-layered silica particle in multiple-ion distribution system.

The Eu(III) distribution mechanism in single extractant-impregnated polymer-layered silica particle in a complex solution containing multiple lanthanide ions was investigated using fluorescence microspectroscopy, which was compared with the single-ion distribution system. The rate-determining step of the Eu(III) distribution was the reaction of Eu(III) with the two extractant molecules in the particle. The distribution mechanism and rate constants obtained in the multiple lanthanide ions-distribution system agreed with those of the single-ion distribution system.

Eu(III) transfer in single N,N,N',N'-tetraoctyldiglycolamide-impregnated polymer-coated silica particle using fluorescence microspectroscopy: transfer mechanism and effect of polymer crosslinking degree.

A microcapillary manipulation system combined with fluorescence microspectroscopy enabled us to analyze mass transfer in a single particle. In this study, we revealed the Eu(III) distribution in a single diglycolamide-derivative extractant (TODGA)-impregnated polymer-coated silica particle. The reaction of Eu(III) with two TODGA molecules in the polymer layer was the rate-limiting process, which was revealed by the relationship between the rate constants (k1 and k-1) and concentrations of Eu(III) and HNO3. The decrease in the crosslinking degree of the polymer layer caused an increase in only k-1. This indicates that hydrophilic environments at lower crosslinking degrees enhance the stability of the charged Eu(III) species such as Eu3+, Eu(NO3)2+, and Eu(NO3)2+.

Pore Size Dependence of Mass Transfer of Zinc Myoglobin in a Single Mesoporous Silica Particle.

This study investigated the pore size dependence of the mass transfer of zinc myoglobin (ZnMb) in a single mesoporous silica particle through confocal fluorescence microspectroscopy. The ZnMb's fluorescence depth profile in the particle was analyzed by a spherical diffusion model, and the intraparticle diffusion coefficient was obtained. The intraparticle diffusion coefficient in the silica particle with various pore sizes (10, 15, 30, and 50 nm) was furthermore analyzed based on a pore and surface diffusion model. Although the mass transfer mechanism in all silica particles followed the pore and surface diffusion model, the adsorption and desorption of ZnMb affected the mass transfer depending on the pore size. The influence of the slow desorption of ZnMb became pronounced for large pore sizes (30 and 50 nm), which was revealed by simulation using a diffusion equation combined with the adsorption-desorption kinetics. The distribution of ZnMb was suppressed in small pore sizes (10 and 15 nm) owing to the adsorption of ZnMb onto the entrance of the pore. Thus, we revealed the mass transfer mechanism of ZnMb in the silica particle with different pore sizes.

Semi-quantification of the binding constant based on bond breaking in a combined acoustic-gravitational field.

In this study, we propose a semi-quantification method based on breaking bonds between microparticles and glass plates in a combined acoustic-gravitational field. The semi-quantified binding constant values for BSA-ibuprofen, BSA-ciprofloxacin, ConA-glycogen, ConA-mannan, and BSA-naproxen calculated using this method were 7.5 × 103, 1.6 × 104, 2.3 × 105, 2.4 × 106 and 9.0 × 107 M-1, respectively, which were in concord with the reported values.

J-aggregation of 5, 10, 15, 20-tetraphenyl-21H, 23H-porphinetetrasulfonic acid in a molecular crowding environment simulated using dextran.

In a molecular crowding environment, different thermodynamics is often observed in a dilute solution. One such example is the promotion of the formation of amyloids, which are causal agents of Alzheimer's disease. Although a considerable number of molecular crowding studies have been reported, its effect remains unclear. In this study, we investigated a J-aggregation of a porphyrin derivative, 5, 10, 15, 20-tetraphenyl-21H,23H-porphinetetrasulfonic acid (TPPS), in a molecular crowding environment simulated by dextran (Dex) in HClO4, HCl, and NaCl solutions. The changes in the number of monomers in the J-aggregate (n) with the concentration of Dex (CDex) depended on the type of solution. No change in n was observed in the NaCl solution, which indicated that the Dex solution did not affect the J-aggregation because of the ionic strength effect. In the HCl solution, the aggregation behavior changed with the pH. Further, at a low pH, the electrostatic interactions promoted J-aggregation by the volume exclusion of Dex, while the aggregation was suppressed at a high pH owing to steric hindrance. A different aggregation mechanism, involving the hydrogen bonding between NH in the center of the TPPS macrocyclic frame and the SO3H and ClO4- functional groups, was responsible for the J-aggregation in the HClO4 solution. Moreover, the n value increased owing to the volume exclusion effect. We expect that this study will be useful for further elucidation of the molecular crowding effect.

Heme-bound tyrosine vibrations in hemoglobin M: Resonance Raman, crystallography, and DFT calculation.

Hemoglobins M (Hbs M) are human hemoglobin variants in which either the α or ß subunit contains a ferric heme in the α2ß2 tetramer. Though the ferric subunit cannot bind O2, it regulates O2 affinity of its counterpart ferrous subunit. We have investigated resonance Raman spectra of two Hbs, M Iwate (α87His → tyrosine [Tyr]) and M Boston (α58His → Tyr), having tyrosine as a heme axial ligand at proximal and distal positions, respectively, that exhibit unassigned resonance Raman bands arising from ferric (not ferrous) hemes at 899 and 876 cm-1. Our quantum chemical calculations using density functional theory on Fe-porphyrin models with p-cresol and/or 4-methylimidazole showed that the unassigned bands correspond to the breathing-like modes of Fe3+-bound Tyr and are sensitive to the Fe-O-C(Tyr) angle. Based on the frequencies of the Raman bands, the Fe-O-C(Tyr) angles of Hbs M Iwate and M Boston were predicted to be 153.5° and 129.2°, respectively. Consistent with this prediction, x-ray crystallographic analysis showed that the Fe-O-C(Tyr) angles of Hbs M Iwate and M Boston in the T quaternary structure were 153.6° and 134.6°, respectively. It also showed a similar Fe-O bond length (1.96 and 1.97 Å) and different tilting angles.

Distribution Behavior of Single-Stranded DNA Molecules in an Amino-Group-Functionalized Silica Microparticle.

In this study, we investigated the distribution behavior of single-stranded DNA molecules with 20 bases in silica particles (particle size: ∼30 µm) using confocal fluorescence microspectroscopy. The distribution kinetics was investigated under various conditions, such as the type of base (adenine, thymine, guanine, and cytosine), pore size of the particle (30 and 50 nm), and salt concentration (100, 200, and 500 mM), which changed the distribution behavior. At high salt concentrations, we observed sigmoidal kinetic behavior, which does not occur in the general distribution of small organic molecules but is often observed in protein aggregation and nuclear growth. An analytical model based on DNA aggregation explained the sigmoidal distribution behavior well, and this model also worked well when the number of DNA molecules involved in DNA aggregation was greater than two. The intraparticle diffusion of DNA molecules was analyzed using the pore and surface diffusion model. As a result, the intraparticle diffusion of DNA aggregates mainly occurs according to surface diffusion, and the surface diffusion coefficient has the same value ((2.4-6.7) × 10-9 cm2 s-1) independent of the pore size and type of base.

Kinetics and mechanism of Eu(III) transfer in tributyl phosphate microdroplet/HNO3 aqueous solution system revealed by fluorescence microspectroscopy.

In this study, we reveal an Eu(III) extraction mechanism at the interface between HNO3 and tributyl phosphate (TBP) solutions using fluorescence microspectroscopy. The mass transfer rate constant at the interface is obtained from the analysis of fluorescence intensity changes during the forward and backward extractions at various HNO3 and TBP concentrations to investigate the reaction mechanism. This result indicates that one nitrate ion reacts with Eu(III) at the interface, whereas TBP molecules are not involved in the interfacial reaction, which is different from the results obtained using the NaNO3 solution in our previous study. We demonstrate that the chemical species of Eu(III) complex with nitrate ion and TBP in the aqueous solution play an important role for the extraction mechanism. The rate constants of the interfacial reactions in the forward and backward extractions are (4.0-5.0) × 10-7 m M-1 s-1 and (3.2-3.3) × 10-6 m s-1, respectively. We expect that our revealed mechanism provides useful and fundamental knowledge for actual solvent extraction.

Direct Quantification of Proteins Modified on a Polystyrene Microparticle Surface Based on ζ Potential Change.

The zeta potential (ζ) of a particle is a surface charge density (σ)-dependent parameter. If a change in σ can be induced by surface modification, the number of molecules modified on the particle can be detected as a measurable change in ζ. In this study, we demonstrate protein detection at zmol or pM levels (bovine serum albumin (BSA), myoglobin (Mb), and lysozyme (Lyz)) on carboxy-functionalized polystyrene (PS) microparticles using the ζ change. Protein modification of the PS particles changes σ because the negatively charged carboxy group is used for protein binding, and proteins also have charged amino acids. The pH dependence of ζ for the protein-modified particles at 4 < pH < 10 is well-explained using the acid dissociation of the acidic and basic amino acids and the Gouy-Chapman-Stern model. An increase in the binding number of proteins per single PS particle (npro/PS) leads to a decrease in ζ, which is consistent with the results estimated by the proposed model. The detection limits of nBSA/PS, nMb/PS, and nLyz/PS are 1.17 × 104, 1.22 × 104, and 1.20 × 104 at pH 8.52, respectively, which means that the concentration-based detection limits are 722, 376, and 371 pM, respectively. We expect that the present method will be a strategy for the detection of molecules on particles.

Zeptomole detection of DNA based on microparticle dissociation from a glass plate in a combined acoustic-gravitational field.

In this study, we propose a novel detection principle based on the dissociation of microparticles immobilized on a glass plate through weak hybridization involving 4-6 base pairs (bps) in a combined acoustic-gravitational field. Particle dissociation from the glass plate occurs when the resultant of the acoustic radiation force (Fac) and the sedimentation force (Fsed) exerted on the particle exceeds the binding force owing to the weak hybridization (Fbind). Because Fac and Fsed can be controlled by the microparticle density, and Fac is a function of the applied voltage to the transducer (V), an increase in V induces particle dissociation. The binding of gold nanoparticles (AuNPs) onto silica microparticles (SPs) resulting from the strong hybridization of 20 bps induces an increase in the density of SPs, leading to an increase in Fac and Fsed; consequently, the voltage V required for dissociation becomes lower than that required without AuNP binding. We demonstrate that the dependence of the binding number of AuNPs per SP on V follows the theoretical prediction. The binding of 7500 AuNPs per SP can be detected as a 10 V change in V. The present approach allows the detection of 2000 DNA molecules involved in the strong hybridization between AuNPs and SP.

Kinetic Analysis of the Mass Transfer of Zinc Myoglobin in a Single Mesoporous Silica Particle by Confocal Fluorescence Microspectroscopy.

The adsorption/desorption mechanisms of biomolecules in porous materials have attracted significant attention because of their applications in many fields, including environmental, medical, and industrial sciences. Here, we employ confocal fluorescence microspectroscopy to reveal the diffusion behavior of zinc myoglobin (ZnMb, 4.4 nm × 4.4 nm × 2.5 nm) as a spherical protein in a single mesoporous silica particle (pore size of 15 nm). The measurement of the time course of the fluorescence depth profile of the particle reveals that intraparticle diffusion is the rate-limiting process of ZnMb in the particle. The diffusion coefficients of ZnMb in the particle for the distribution (Ddis) and release (Dre) processes are determined from the rate constants, e.g., Ddis = 1.65 × 10-10 cm2 s-1 and Dre = 3.68 × 10-10 cm2 s-1, for a 10 mM buffer solution. The obtained D values for various buffer concentrations are analyzed using the pore and surface diffusion model. Although surface diffusion is the main distribution process, the release process involves pore and surface diffusion, which have not been observed with small organic molecules; the mechanism of transfer of small molecules is pore diffusion alone. We demonstrate that the mass transfer kinetics of ZnMb in the silica particle can be explained well on the basis of pore and surface diffusion.

Effect of Molecular Crowding on Complexation of Metal Ions and 8-Quinolinol-5-Sulfonic Acid.

The effect of molecular crowding on macromolecular reactions has been revealed by many researchers. In this study, we investigate the complexation of metal ions (Zn, Co, and Cd) with 8-quinolinol-5-sulfonic acid as a model of small-molecular reactions in molecular crowding. The complexation constants for 1:1, 1:2, and total complexation in the presence of polyethylene glycol (PEG, a molecular crowding reagent) are evaluated based on the increase in the reactant activity by volume exclusion and the decrease in the water activity due to the change in osmotic pressure. All complexation constants are enhanced by increasing the concentration of PEG. Its mechanisms differ for 1:1, 1:2, and total complexation. The 1:1 complexation is promoted only by the influence of the water activity, while the reactant and water activities influence the increase in the 1:2 complexation constant. Increasing the molecular weight of PEG further increases the complexation constants, as dehydration of the complex is promoted by a higher hydration number of PEG. Because this study gives the fundamental knowledge for the protein-metal interaction, in which solvation is an important factor, in molecular crowding, it provides new insights into molecular crowding studies and should attract the attention of a broad spectrum of biochemistry researchers.

Three-dimensional graphing representing six variables for speed and separation performance in liquid chromatography.

The two variables, flow rate and column length, enable naive determination of the number of theoretical plates (N) in isocratic elution; this, in turn, enables the formation of a three-dimensional graph with N as the z-axis. An alternate three-dimensional graph with N as the z-axis can be drawn, then, with the alternate basal plane illustrating the pressure drop and hold-up time. In this article, the pressure drop and hold-up time are formulated so as to be represented unitarily in the former graph, because the flow rate and column length interact simultaneously as operational variables. This formulation manipulates both the pressure drop and the hold-up time as logarithmic axes, to evaluate the landscape. Also of use is the representation, in the same graph, of the height equivalent to a theoretical plate, as the fundamental property of the packing supports. For this purpose, the number of theoretical plates per unit length are here introduced as the sixth variable, instead of the height equivalent to a theoretical plate. Representing the six variables in three-dimensional graphs enables a clear understanding both of the separation condition optimization methods and the relation among variables for the speed and separation performance. The linear velocity, column length, N, velocity-length product, hold-up time, and number of theoretical plates per unit length, are here selected as the six elementary variables for the three-dimensional graphs; and, based on the packing supports of 2, 3, and 5-µm particle and monolithic columns. Finally, the usage of logarithmic three-dimensional graph is illustrated for understanding the speed and separation performance.

Three approaches to improving performance of liquid chromatography using contour maps with pressure, time, and number of theoretical plates.

Attempts to improve HPLC performance often focus on increasing the speed or separation performance. In this article, both the flow rate and column length are optimized as separation conditions, while observing the number of theoretical plates and hold-up time with isocratic elutions. In addition, the upper pressure limit must be simultaneously considered as the boundary condition. Approaches based on the optimal velocity (Opt.) are often adopted; but the kinetic performance limit (KPL) in Desmet's method can also be utilized for three-dimensional graphing with axes of pressure, time, and number of theoretical plates. Here, two approaches involving pressure increase are introduced, beginning with the condition of optimal linear velocity: one aimed at greater speed and the other at higher resolution. Coefficients of pressure-application are derived to measure the effectiveness of the intermediate conditions between the Opt. and KPL methods. In the third approach, the hold-up time is extended while maintaining a fixed pressure. Coefficients of time-extension are also derived, to determine the effectiveness to improve the separation performance.