Hydrophilic Probe Research Articles

Probing Electrostatic and Hydrophobic Associative Interactions in Cells.

Weak nonspecific interactions between biomacromolecules determine the cytoplasmic organization. Despite their importance, it is challenging to determine these interactions in the intracellular dense and heterogeneous mixture of biomacromolecules. Here, we develop a method to indicate electrostatic and hydrophobic associative interactions and map these interactions. The method relies on a genetically encoded probe containing a sensing peptide and a circularly permuted green fluorescent protein that provides a ratiometric readout. Inside bacterial and mammalian cells, we see that the cytoplasmic components interact strongly with cationic and hydrophobic probes but not with neutral hydrophilic probes, which remain inert. The Escherichia coli cytoplasm interacts strongly with highly negatively charged hydrophilic probes, but the HEK293T cytoplasm does not. These associative interactions are modulated by ATP depletion. Hence, the nonspecific associative interaction profile in cells is condition- and species-dependent.

Shear viscosity of a near-critical binary system analyzed with variable-temperature atomic force microscopy

Variation in shear viscous drag in a near-critical binary mixture was measured between a hydrophilic probe sphere and a hydrophilic substrate down to a distance range comparable to or smaller than the correlation length ξ using a magnetic excitation method of atomic force microscopy at temperatures T=Tc−∆T with ∆T varying from 0.1 to 0.7 K. As the temperature approached the Tc, the increase in the drag coefficient toward the zero distance was observed to be less pronounced in the distance range below ca. 100 nm. The data was discussed as arising from suppression in a concentration gradient in the intersurface region due to the overlap of the affected layers on similar surfaces.

Screening, Construction, and Preliminary Evaluation of CLDN18.2-Specific Peptides for Noninvasive Molecular Imaging.

Recent global clinical trials have shown that CLDN18.2 is an ideal target for the treatment of gastric cancer and that patients with high CLDN18.2 expression can benefit from targeted therapy. Therefore, accurate and comprehensive detection of CLDN18.2 expression is important for patient screening and guidance in anti-CLDN18.2 therapy. Phage display technology was used to screen CLDN18.2-specific peptides from 100 billion libraries. 293TCLDN18.1 cells were used to exclude nonspecific binding and CLDN18.1 binding sequences, while 293TCLDN18.2 cells were used to screen CLDN18.2-specific binding peptides. The monoclonal clones obtained from phage screening were sequenced, and peptides were synthesized based on the sequencing results. Binding specificity and affinity were assessed with a fluorescein isothiocyanate (FITC)-conjugated peptide. A 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated peptide was also synthesized for 68Ga radiolabeling. The in vitro and in vivo stability, partition coefficients, in vivo molecular imaging, and biodistribution were also characterized. Overall, 54 monoclonal clones were selected after phage display screening. Subsequently, based on the cell ELISA results, CLDN18.2 preference monoclonal clones were selected for deoxyribonucleic acid (DNA) sequencing, and four 7-peptide sequences were obtained after sequence comparison; among them, a peptide named T37 was further validated in vitro and in vivo. The T37 peptide specifically recognized CLDN18.2 but not CLDN18.1 and bound strongly to CLDN18.2-positive cell membranes. The 68Ga-DOTA-T37 probe exhibits good in vitro properties and high stability as a hydrophilic probe; it has high biological safety, and positron emission tomography/computed tomography (PET/CT) studies have shown that it can specifically target CLDN18.2 protein and CLDN18.2-positive tumors in mice. 68Ga-DOTA-T37 demonstrated the superiority and feasibility of using a CLDN18.2-specific probe in PCT/CT imaging, which deserves further development and exploitation.

Click postsynthesis of microporous organic network@silica composites for reversed-phase/hydrophilic interaction mixed-mode chromatography.

Recently, the good physical and chemical properties, well-defined pore architectures, and designable topologies have made microporous organic networks (MONs) excellent potential candidates in high-performance liquid chromatography (HPLC). However, their superior hydrophobic structures restrict their application in the reversed-phase mode. To solve this obstacle and to expand the application of MONs in HPLC, we realized the thiol-yne "click" postsynthesis of a novel hydrophilic MON-2COOH@SiO2-MER (MER denotes mercaptosuccinic acid) microsphere for reversed-phase/hydrophilic interaction mixed-mode chromatography. SiO2 was initially decorated with MON-2COOH using 2,5-dibromoterephthalic acid and tetrakis(4-ethynylphenyl)methane as monomers, and MER was then grafted via thiol-yne click reaction to yield MON-2COOH@SiO2-MER microspheres (5μm) with a pore size of ~1.3nm. The -COOH groups in 2,5-dibromoterephthalic acid and the post-modified MER molecules considerably improved the hydrophilicity of pristine MON and enhanced the hydrophilic interactions between the stationary phase and analytes. The retention mechanisms of the MON-2COOH@SiO2-MER packed column were fully discussed with diverse hydrophobic and hydrophilic probes. Benefiting from the numerous -COOH recognition sites and benzene rings within MON-2COOH@SiO2-MER, the packed column exhibited good resolution for the separation of sulfonamides, deoxynucleosides, alkaloids, and endocrine-disrupting chemicals. A column efficiency of 27,556 plates per meter was obtained for the separation of gastrodin. The separation performance of the MON-2COOH@SiO2-MER packed column was also demonstrated by comparing with those of MON-2COOH@SiO2, commercial C18, ZIC-HILIC, and bare SiO2 columns. This work highlights the good potential of the thiol-yne click postsynthesis strategy to construct MON-based stationary phases for mixed-mode chromatography.

Solution Structure and Hydration Forces between Mica and Hydrophilic Versus Hydrophobic Surfaces

Solid–liquid interfaces are central to a range of interesting phenomena including colloidal aggregation, crystallization by particle attachment, catalysis, heterogeneous nucleation, water desalination, and biomolecular assembly. While three-dimensional atomic force microscopy (3D AFM) has emerged as a technique for resolving interfacial solution structure at the molecular scale, key challenges for data interpretation persist, most notably regarding the influence of the probe on the measured structure. Using the mica–water system as a case study, we investigate the effect of hydrophilic and hydrophobic probes on interfacial solution structure measured by 3D AFM. Data from hydrophilic silicon-based probes are in good agreement with molecular dynamics simulations, wherein the innermost water molecules adsorb preferentially at the surface ditrigonal cavity sites, followed by two additional ordered hydration layers. In contrast, the hydrophobic carbon-based probes detect vertical oscillatory features but do not show lateral patterning that matches the underlying mica lattice. At high ionic strength, up to six of these oscillatory features are observed extending 2 nm into the solution phase with an average spacing of 0.29 ± (0.04) nm. We also determine that the repulsive hydration force between mica and the hydrophilic probe depends on the nature and concentration of ions in solution. Specifically, solutions with stronger ion–water and ion–ion interactions produce a stronger repulsive hydration force as the probe approaches the surface. Based on these observations, we present a scheme for controlling the outcomes of particle aggregation and attachment by varying the solution conditions to tune the hydration force.

PH-Dependent disruption of giant polymer vesicles: a step towards biomimetic membranes

The spatiotemporal pH-triggered controlled release of a hydrophilic probe in a pH-responsive PGUV system demonstrates its potential as a biomimetic system for drug delivery, microreactors and artificial cell mimics.

Synergistic Effect of Bio-Inspired Nanochannels: Hydrophilic DNA Probes at Inner Wall and Hydrophobic Coating at Outer Surface for Highly Sensitive Detection.

During the past few decades, bio-inspired nanochannels have been well developed and applied in biosensing, energy transfer, separation, and so on. Here, inspired by the synergistic effect of biological nanopores, biomimetic solid-state nanochannels with hydrophilic DNA probes at the inner wall (DNA@IWHydrophilic ) and hydrophobic coating at the outer surface (None@OSHydrophobic ) are designed. To demonstrate their prompted sensing properties, Hg2+ and its specific probe are selected as target and hydrophilic DNA probes, respectively. Compared with the traditional solid-state nanochannels with hydrophilic probes distributed on both the inner wall and outer surface, the nanochannels with DNA@IWHydrophilic +None@OSHydrophobic significantly decrease the limit of detection (LOD) by 105 -fold. The obvious improvement of sensitivity (with LOD of 1nM) is attributed to the synergistic effect: None@OSHydrophobic results in the nanochannel's effective diameter decrease and DNA@IWHydrophilic induces a specific sensing target. Meanwhile, nanomolar detection of Hg2+ in human serum and in vivo fish muscle are achieved. Through molecular dynamics simulation, the synergistic effect can be confirmed by ion fluxes increasement;the relative carbon nanotube increases from 135.64% to 135.84%. This work improves the understanding of nanochannels' synergistic effect and provides a significant insight for nanochannels with improved sensitivity.

Monomer-mediated fabrication of microporous organic network@silica microsphere for reversed-phase/hydrophilic interaction mixed-mode chromatography

Microporous organic networks (MONs) are promising in high performance liquid chromatography (HPLC) with large specific surface area, good hydrophobicity and stability. However, their superhydrophobic structures restrict MONs-based HPLC only in reversed-phase mode. To decrease the hydrophobicity of pristine MONs and to expand their broad application in HPLC, here we described the monomer-mediated fabrication of core-shell MON-2COOH@SiO2 microspheres for reversed-phase liquid chromatography (RPLC)/hydrophilic interaction liquid chromatography (HILIC) mixed-mode chromatography for the first time. The –COOH groups were introduced into MONs’ skeleton to improve their hydrophilicity and to provide hydrophilic interaction sites. The MON-2COOH was grafted onto silica via a monomer mediated method to produce monodispersed core-shell microspheres. By adjusting the concentration of reactants, the thickness of MON-2COOH shell was easily manipulated. The packed MON-2COOH@SiO2 column showed high resolution and selectivity for separating both hydrophobic (alkylbenzenes, polycyclic aromatic hydrocarbons, anilines and phenols) and hydrophilic (nucleoside and nucleic bases) probes, highlighting the promise of MONs in mixed-mode HPLC. The MON-2COOH@SiO2 column also achieved good separation to sulfonamides, nonsteroidal anti-inflammatory drugs, flavonoids and phenylurea herbicides, and offered better resolution than commercial C18 and pristine SiO2 column. Multiple retention mechanisms were also found on MON-2COOH@SiO2 packed column, underlining the great potential of MONs in mixed-mode HPLC.

Bi-amino acid functionalized biomimetic honeycomb chitosan membrane as a multifunctional hydrophilic probe for specific capture of N-linked glycopeptides in nasopharyngeal carcinoma's disease patient's serum.

In this work, a novel porous bifunctionalized composite material was synthesized via a simple method. Gold nanoparticles are uniformly dispersed on the surface of the biomimetic honeycomb chitosan membrane through the interaction between amino and Au, and then cysteine and glutathione are successfully grafted onto the surface of the Au by the Au-S bond. The modification of cysteine and glutathione makes this bifunctionalized composite material have significant advantages of superhydrophilicity and small steric hindrance simultaneously. This material manifests excellent property in glycopeptides enrichment, with high selectivity (1:5000), low detection limit (0.1fmol·μL-1 ), high recovery rate (99.4 ± 0.5%), and good repeatability. In addition, with the help of nano-flow liquid chromatography tandem mass spectrometry, this composite achieved excellent performance in efficiently enriching glycopeptides in the serum of healthy people and nasopharyngeal carcinoma's disease patient. More excitingly, further gene ontology analysis of molecular function and biological process indicated that 41 original glycoproteins of the identified glycopeptides from serum of nasopharyngeal carcinoma's disease patient significantly partake in numerous cancer-associated events, including protease binding, calcium ion binding, enzyme binding, extracellular matrix organization, cellular response to tumor necrosis factor, and inflammatory response.

A ratiometric electrochemical biosensor for analysis of total N-glycolylneuraminic acid based on pH-adjusted self-assembly of lipid bilayer

In this work, we propose a pH-adjusted self-assembled lipid bilayer (LB) as signal output mode for the analysis of total N-glycolylneuraminic acids (TNeu5Gc) including free Neu5Gc and conjugated Neu5Gc. Based on synergistic inhibition effect of pH-adjusted self-assembled LB and TNeu5Gc on transport of hydrophilic probe, a ratiometric electrochemical biosensor has been elaborately constructed to decrease the experimental interferences and enhance analytical accuracy. The constructed biosensor has been successfully applied for electrochemical analysis of TNeu5Gc with a wide linear detection range from 2.816 ng/mL to 360.396 ng/mL and a low detection limit of 0.506 ng/mL. This work not only firstly establishes a ratiometric electrochemical biosensor for the analysis of TNeu5Gc, but also provides a new insight for self-assembly of LB in response to pH value change.

Advances in Automated Piston Liquid-Liquid Microextraction Technique

A new automated micro liquid-liquid extraction technique was successfully developed. This novel syringe-based technique capitalizes on the advantages of vigorous fluid agitation and the shearing effect of two fluids with different properties to achieve high extraction efficiency. The technique is at least 20 times faster than mechanical shaking or sonication in achieving a similar recovery even with a hydrophilic probe molecule such as 1,4-dioxane in an aqueous medium. Excellent repeatability with a relative standard deviation as low as 0.56% over a five-day test, n = 2 per day, was demonstrated with 1,4-dioxane. Other model compounds in aqueous matrices evaluated, including phenolics and extraction solvents like chloroform and hexane, showed similar performance in repeatability. An added advantage of this technique involves performing multiple extractions. Its capabilities in conducting complicated extraction steps and minimizing the use of organic solvents as low as 200 µL to achieve a preconcentration effect were demonstrated. The technique is suitable for use with emulsion-forming samples without further sample manipulation by incorporating a demulsifier such as acetone during the extraction process. The technique was found to be efficient and environmentally friendly with low solvent waste. This technique is ideal for implementation in automated high throughput and cost-effective quality assurance laboratory environments.

Characteristics of interfacial nanobubbles and their interaction with solid surfaces

For the application development of interfacial nanobubbles (INBs), it is of great significance for understanding the special physicochemical characteristics of INBs and the interactions with hydrophilic and hydrophobic surfaces. In this paper, the morphology of INBs was scanned and the interaction between INBs and different surfaces were researched by atomic force microscope with the prepared hydrophilic/hydrophobic probes. In this investigation, the dynamics of INBs growth was obtained. The hydrophilic probe is essential to scan the complete morphology of INBs. Based on the detailed analysis of the interaction between the INB and the hydrophilic probe, the vertical height of the INB corresponding position can be calculated from the extending force curve. By setting different applied forces, it is found that there is a positive correlation between the INB vertical height and the applied force, that is, the higher position of INB needs the greater applied force to reach the bottom of the INB. For instance, the point a (7.4 nm) and the point d (65.4 nm) require 0.7 nN, 6.5 nN, respectively. The morphology of INBs is seriously deformed with the hydrophobic probe. What is more, there is only one inflection point on the extending force curve so that the vertical height of INBs cannot be calculated. In addition, the interaction between INBs and hydrophobic surface is significant stronger, comparing with hydrophilic surface.

Femtosecond solvation dynamics study of hydrophobic and hydrophilic probes in various room temperature ionic liquids (RTILs) containing microemulsions

The locations of hydrophobic and hydrophilic probes in room temperature ionic liquids (RTILs) containing microemulsions have been investigated using femtosecond solvation dynamics study, as the time correlated single photon counting (TCSPC) measurement has limitation in terms of detecting the ultrafast component of relaxation time less than 100 ps. Here, choice of two different RTILs unveils the information about the effect of acyclic and cyclic hydrocarbon chain on solvation dynamics. The exclusive information from dynamic light scattering (DLS) measurement, anisotropy study and the fluorescence correlation spectroscopy (FCS) judiciously help to perform detailed analysis of these systems.

Bifunctional Therapeutic Application of Low-Frequency Ultrasound Associated with Zinc Phthalocyanine-Loaded Micelles.

PurposeSonodynamic therapy (SDT) is a new therapeutic modality for the noninvasive cancer treatment based on the association of ultrasound and sonosensitizer drugs. Topical SDT requires the development of delivery systems to properly transport the sonosensitizer, such as zinc phthalocyanine (ZnPc), to the skin. In addition, the delivery system itself can participate in sonodynamic events and influence the therapeutic response. This study aimed to develop ZnPc-loaded micelle to evaluate its potential as a topical delivery system and as a cavitational agent for low-frequency ultrasound (LFU) application with the dual purpose of promoting ZnPc skin penetration and generating reactive oxygen species (ROS) for SDT.MethodsZnPc-loaded micelles were developed by the thin-film hydration method and optimized using the Quality by Design approach. Micelles’ influence on LFU-induced cavitation activity was measured by potassium iodide dosimeter and aluminum foil pits experiments. In vitro skin penetration of ZnPc was assessed after pretreatment of the skin with LFU and simultaneous LFU treatment using ZnPc-loaded micelles as coupling media followed by 6 h of passive permeation of ZnPc-loaded micelles. The singlet oxygen generation by LFU irradiation of the micelles was evaluated using two different hydrophilic probes. The lipid peroxidation of the skin was estimated using the malondialdehyde assay after skin treatment with simultaneous LFU using ZnPc-loaded micelles. The viability of the B16F10 melanoma cell line was evaluated using resazurin after treatment with different concentrations of ZnPc-loaded micelles irradiated or not with LFU.ResultsThe micelles increased the solubility of ZnPc and augmented the LFU-induced cavitation activity in two times compared to water. After 6 h ZnPc-loaded micelles skin permeation, simultaneous LFU treatment increased the amount of ZnPc in the dermis by more than 40 times, when compared to non-LFU-mediated treatment, and by almost 5 times, when compared to LFU pretreatment protocol. The LFU irradiation of micelles induced the generation of singlet oxygen, and the lipoperoxidation of the skin treated with the simultaneous LFU was enhanced in three times in comparison to the non-LFU-treated skin. A significant reduction in cell viability following treatment with ZnPc-loaded micelles and LFU was observed compared to blank micelles and non-LFU-treated control groups.ConclusionLFU-irradiated mice can be a potential approach to skin cancer treatment by combining the functions of increasing drug penetration and ROS generation required for SDT.

Study of Interactions between Interfacial Nanobubbles and Probes of Different Hydrophobicities.

In this study, hydrophilic, medium hydrophobic, and strong hydrophobic probes are obtained via treatment with plasma and octadecyl trichlorosilane. The interaction between the probes and interfacial nanobubbles (INBs) is examined using atomic force microscopy. The results show that a hydrophilic probe can scan the true shape of the INBs, and the distance between the first inflection point and the zero point of the approach force curve is equal to the vertical height of the nanobubble. The medium hydrophobic probe caused severe deformation of INB morphologies in the horizontal direction during scanning; nevertheless, the complete shape of the INB is obtained using this probe by lowering the scanning parameters. However, the characteristic of the approach force curve proves that the size of the nanobubbles is underestimated. The strong hydrophobic probe deforms INB morphologies severely, whose size cannot be obtained. The maximum attractive force in the approach force curve and the adhesive force in the retract force curve obtained using the strong hydrophobic probe are approximately 6 and 12 nN, respectively, which are both higher than those of the hydrophilic and medium hydrophobic probes. It is reasoned that the liquid film is maintained between the hydrophilic probe and the INBs, the medium hydrophobic probe pierces the INBs slightly, while the strong hydrophobic probe punctures the liquid film and demonstrates a pinning effect.

Solvent Effects on Skin Penetration and Spatial Distribution of the Hydrophilic Nitroxide Spin Probe PCA Investigated by EPR

Oxidative stress occurs in extrinsic skin aging processes and diseases when the enhanced production of free radicals exceeds the homeostatic antioxidant capacity of the skin. The spin probe, 3-(carboxy)-2,2,5,5-tetramethylpyrrolidin-1-oxyl (PCA), is frequently used to study the cutaneous radical production by electron paramagnetic resonance (EPR) spectroscopy. This approach requires delivering PCA into the skin, yet solvent effects on the skin penetration and spatial distribution of PCA have not been thoroughly investigated. Three solvents of ethanol, phosphate-buffered saline (PBS) and ethanol-PBS (1:1) were studied. For both human and porcine skin ex vivo, the amount of PCA in the stratum corneum (SC) was the lowest when using ethanol and very similar for PBS and ethanol-PBS. The highest amount of PCA in the viable skin layers was detected for ethanol-PBS, yet it only took up less than 5% of the total amount. The majority of PCA was localized in the SC, among which PCA with high mobility was predominantly distributed in the hydrophilic microenvironment of corneocytes and PCA with lower mobility was mainly in the less hydrophilic microenvironment of intercellular skin lipids. A higher ethanol concentration in the solvent could improve the distribution of PCA in the hydrophilic microenvironments of the SC. The results suggest that ethanol-PBS (1:1) is best-suited for delivering most PCA deep into the skin. This work enhances the understanding of solvent effects on the skin penetration and distribution of PCA and supports the utilization of PCA in studying cutaneous radical production.

Nanoconfinement Effects on Redox Probe Transport in Lipid Assemblies on and in Mesoporous Silica Thin Films

AbstractRedox probe transport through supported lipid bilayers and nanopore‐confined lipid assemblies on silica thin films is examined using electrochemical impedance spectroscopy (EIS). These supported lipid systems are emerging biomimetic separation and sensor platforms. The ability to quantify the accessibility of the pore structure of the mesoporous silica thin films is demonstrated, which is essential for the incorporation of carriers into the lipids for selective solute transport. Redox probe molecules with varying hydrophilicity are used to compare ion transport in supported lipid pore‐spanning bilayers (enveloped bilayers) and novel lipid filled pores of mesoporous silica thin films. The films feature orthogonally oriented 8–10 nm cylindrical nanopores formed by deposition of P123‐templated silica sols onto chemically modified fluorine‐doped tin oxide. Nanopore accessibility is confirmed by EIS with hydrophilic probe 1,1′‐ferrocenedimethanol (FDM). Filling the pores with lipid 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine results in a superior barrier (with roughly 1/9 the permeability) to transport of FDM compared to fragile enveloped lipid bilayers deposited by vesicle fusion. The pore‐confined lipids not only provide a better barrier to FDM, but also a better pathway for the transport across the films of a hydrophobic redox probe 1,1′‐dioctadecyl‐4,4′‐bipyridinium dibromide, with an ideal transport selectivity of 11 compared to FDM.

Hydrophilic Fluorescent Probes for Fe3+ Ions Based on Nanoparticles of Twisting D-π-A Type Compound Derived from Benzylidenemalononitrile

Hydrophilic Fluorescent Probes for Fe³⁺ Ions Based on Nanoparticles of Twisting D-π-A Type Compound Derived from Benzylidenemalononitrile

Hydrophilic Silver Nanoparticles Loaded into Niosomes: Physical-Chemical Characterization in View of Biological Applications.

Silver nanoparticles (AgNPs) are widely used as antibacterial agents and anticancer drugs, but often their low stability limits their mass production and broad applications. The use of niosomes as a carrier to protect and envelop AgNPs gives a new perspective to solve these problems. In this study, AgNPs were functionalized with sodium 3-mercapto-1-propanesulfonate (3MPS) to induce hydrophilic behavior, improving loading in Tween 20 and Span 20 niosomes (NioTw20 and NioSp20, respectively). Entrapment efficiency was evaluated by UV analyses and is around 1–4%. Dimensions were investigated by means of dynamic light scattering (DLS) (<2RH> = 140 ± 4 nm and <2RH> = 251 ± 1 nm respectively for NioTw20 + AgNPs and NioSp20 + AgNPs) and were compared with those by atomic force microscopy (AFM) and small angle X ray scattering (SAXS) analyses. Stability was assessed in water up to 90 days, and both in bovine serum and human serum for up to 8 h. In order to characterize the local structure of niosomes, SAXS measurements have been performed on Tween 20 and Span 20 empty niosomes and loaded with AgNPs. The release profiles of hydrophilic probe calcein and lipophilic probe Nile Red were performed in HEPES buffer and in human serum. All these features contribute to conclude that the two systems, NioTw20 + AgNPs and NioSp20 + AgNPs, are suitable and promising in the field of biological applications.

Are water-xylitol mixtures heterogeneous? An investigation employing composition and temperature dependent dielectric relaxation and time-resolved fluorescence measurements

Aqueous xylitol solutions at six different concentrations were studied employing dielectric relaxation (DR) and time-resolved fluorescence (TRF) measurements in the temperature range 295–323 K. The focus was to explore the solution heterogeneity aspect via monitoring the viscosity coupling of the average relaxation rates at various temperatures. TRF measurements were done using both hydrophobic and hydrophilic probes to explore the preferences, if any, for solute locations in these binary mixtures. Energy-selective population excitations and the corresponding fluorescence emissions did not suggest any significant spatial heterogeneity in solution structure within the lifetimes of these probes. DR measurements and TRF experiments indicated mild deviations from the hydrodynamic viscosity dependence of the measured relaxation rates. All these suggest mild spatiotemporal heterogeneity for these water-xylitol mixtures in the temperature range considered. In addition, DR timescales appear to originate from reorientational and H-bond relaxation dynamics, excluding the possibility of full molecular rotations. Heterogeneity in water-xylitol mixtures was investigated employing dielectric relaxation (DR) and Time-resolved fluorescence (TRF) spectroscopic techniques. Time-resolved anisotropy and DR response were found to depend both on xylitol concentration and temperature. Signature of mild heterogeneity was detected along with presence of water molecules that were slower than bulk-like water. Slower than bulk DR response vanishes with an increase of solution temperature.