Cryoprotectants and dynamics of exclusion zone water.

T1-weighted magnetic resonance images of water in the surroundings of a Nafion surface allowed the identification of the presence of a low-mobility zone (LMZ), 60 μm thick, consisting of water molecules structured in a hydrogen-bonding network, promoted by the presence of the acidic protons on the surface of the sulphonated polymer. In parallel, the exclusion zone (EZ) was assessed by observing in optical microscopy the distribution of microspheres suspended in the medium in contact with the Nafion membrane. It was found that the LMZ and the EZ do not correspond: in fact, the former is thinner and more stable over time than the latter and they behave differently when ions are present in the medium in which Nafion is immersed.

Long-range interactions keep bacterial cells from liquid-solid interfaces: Evidence of a bacteria exclusion zone near Nafion surfaces and possible implications for bacterial attachment

Exclusion zone (EZ) formation at water-membrane interfaces was studied via bright- and dark-field microscopy. Various aqueous colloids including suspensions of charged microspheres, silicon dioxide particles, and raw whole milk were studied with Nafion® hydrophilic membranes. Interfacial formations observed included EZs and more complex patterns including striations, double layers, banding, dendritic aggregates of particles, and double-stranded structures resembling Birkeland current filaments in cold plasmas. A complex three-dimensional dynamic structure and continuous flow patterns persist in and around EZs, maintaining movement of the colloidal particles even after EZs are fully formed, for which a schematic is proposed. Since radiant energy is critical for EZ formation, we hypothesize that these interfacial phenomena are non-equilibrium dissipative structures that self-organize and self-maintain due to ongoing dynamic processes that may involve hydrodynamic interactions. Another experimental approach undertaken involved the construction of a microscope flow cell to measure the kinetics of EZ formation using sequential microphotography analyzed with macro-programmed ImageJ software to investigate effects of different types of conditioned water. No significant difference was found between spring water and the same water treated by a magnetic vortexer. A significant difference was found for municipal tap water compared to electrolyzed alkaline tap water from the same source.

Many hydrophilic materials in aqueous solution show near-surface zones that exclude suspended colloids and dissolved molecules. These exclusion zones (EZs) can extend for tens to hundreds of micrometers from the hydrophilic surface, and show physicochemical properties that differ from bulk water. So far, only continuous surfaces of polymers, gels, or biological specimens have been studied. In this report, we explore the interactions between exclusion zones generated by discontinuous, regularly spaced EZ-generating surfaces, namely strips of Nafion on a glass surface. Various inter-strip spacings were studied. When Nafion surfaces are separated by 100 micrometers or less, EZs merged with one another, forming a single, continuous, stable EZ. Separations larger than 100 micrometers produced discontinuous EZs that did not merge. This result has implication for the mechanism by which independent EZs can merge with one another.

BackgroundWater undergoes structural change as it interfaces with hydrophilic surfaces, including the many hydrophilic surfaces within the cell. This interfacial water has become known as “Exclusion Zone (EZ) water” or “fourth-phase water” [1].MethodsWe tested the hypothesis that anesthetics diminish the amount of EZ water, and that this change may correlate with functional changes in anesthesia. By using the local anesthetics Lidocaine and Bupivacaine as well as a general inhalational anesthetic, Isoflurane, we tracked the EZ size as these anesthetics were introduced.ResultsAll three anesthetics diminished EZ size in a concentration-dependent manner at concentrations of 0.18 mM and greater for Bupivacaine, 0.85 mM and greater for Lidocaine, and 0.2% for Isoflurane. At extremely low (micromolar) concentrations, however, all three anesthetics increased EZ size.ConclusionsThe sharp increase of EZ size associated with micromolar anesthetic concentrations follows a similar pattern to induction of general anesthesia, from the excitation stage (Stage II) to the depression and overdose stages of surgical anesthesia (Stages III and IV). The results are consistent with the hypothesis that anesthetics may act on water, a fundamental organizational component common to all cells.

Interfacial water lying next to hydrophilic surfaces has been shown to be spectroscopically, mechanically, and electrically distinct from bulk water. Interfacial water has also been shown to exclude negatively and positively charged microspheres and has thus become known as the "exclusion zone". Measurements have demonstrated that exclusion zones exhibit a negative electrical potential on the order of -100 mV relative to bulk water, with a corresponding distribution of positive protons in the bulk water region beyond the exclusion zone. This separation of charge is hypothesized to create an electrostatic force between the exclusion zone and the proton-enriched zone beyond. To test this hypothesis, a hydrophilic Nafion ring was attached to the tip of a deflectable ribbonlike force sensor. The sensor was designed to obstruct the flow of protons from one side of the lever to the other, so that any proton-based force would remain unilateral. pH-sensitive dye measurements confirmed that the protons were largely confined to one side. When the lever assembly was exposed to water, the sensor deflected toward the protons. Over a period of 20 min, deflection amounted to approximately 20 μm, corresponding to a force of approximately 22 μN. Hence, electrostatic forces are confirmed. If exclusion zones exist ubiquitously at hydrophilic surfaces, including biological surfaces, then the resulting electrostatic forces may play significant roles in many biological phenomena including adhesion and protein folding.

Effect of infrared radiation on interfacial water at hydrophilic surfaces

To present clinical and experimental evidence of the role of exclusion zone (EZ) water in photobiomodulation. Water at the interface of most hydrophilic surfaces forms a solute-free area, or EZ, that can project for hundreds of microns. To date, EZ phenomena had been documented in nafion, resins, and biologic membranes. Retrospective analysis of published experimental and clinical data using an infrared pulsed laser device (IPLD). Photo-induced effects on the water dynamics of burned rat tissue monitored by 1H-NMR transverse relaxation times (1/T2) indicate significantly greater structuring of water. A microdensitometry study of T2 weighted tumor heterogeneities from a phase I clinical trial in patients with advanced neoplasias and an algorithm for tumor characterization also shows significantly increased structuring of water associated with biopolymers and macromolecules. To the best of our knowledge, this is the first known demonstration of the EZ in medicine. Data support the premise that photobiomodulation can increase potential energy in the EZ, which then acts as an energy repository that can selectively supplement cell energy demands. It further suggests EZ structuring may be used as a predicator of anticancer response before measurable tumor volume reduction.

While recent research on interfacial water has focused mainly on the few interfacial layers adjacent to the solid boundary, century-old studies have extensively shown that macroscopic domains of liquids near interfaces acquire features different from the bulk. Interest in these long-range effects has been rekindled by recent observations showing that colloidal and molecular solutes are excluded from extensive regions next to many hydrophilic surfaces [Zheng and Pollack Phys. Rev. E 2003, 68, 031408]. Studies of these aqueous "exclusion zones" reveal a more ordered phase than bulk water, with local charge separation between the exclusion zones and the regions beyond [Zheng et al. Colloid Interface Sci. 2006, 127, 19; Zheng and Pollack Water and the Cell: Solute exclusion and potential distribution near hydrophilic surfaces; Springer: Netherlands, 2006; pp 165-174], here confirmed using pH measurements. The main question, however, is where the energy for building these charged, low-entropy zones might come from. It is shown that radiant energy profoundly expands these zones in a reversible, wavelength-dependent manner. It appears that incident radiant energy may be stored in the water as entropy loss and charge separation.

Brownian particles suspended in water or other polar liquids are pushed out of the region next to hydrophilic polymers, leaving a microsphere-free region known as the “exclusion zone” (EZ). This study aimed to test the hypothesis that the dilution of ethanol in water may influence EZ formation. EZs were created in aqueous media using Nafion tubes as EZ-nucleating surfaces. To define the outer edge of the EZ, carboxylate microspheres, 1 µm diameter, were used. Dynamic movement of microspheres away from Nafion surface was registered in mixtures of ethanol and water, the ethanol concentration varying from 0 to 95%. We found that mixtures with the highest concentrations of ethanol generally produced the smallest EZs and the slowest EZ buildup. However, an unexpected result was the presence of an extremum corresponding to ~10% ethanol. At this concentration, the EZ is larger than in either pure water or almost pure ethanol.

Water molecules play critical roles in many biological functions, such as protein dynamics, enzymatic activities, and cellular responses. Previous nuclear magnetic resonance and neutron scattering studies have shown that water molecules bind to specific sites on surfaces and form localized clusters. However, most current experimental techniques cannot measure dynamic behaviors of ordered water molecules on cell-size (10μm) scale. Recently, the long-distance effect of structured water has been demonstrated by Pollack and his colleagues. Namely, there is a structured water layer near the hydrophilic surface that can exclude solutes (Zheng et al, Adv Colloid Interface Sci 127:19-27, 2006; Pollack 2006, Adv Colloid Interface Sci 103:173-196, 2003). The repelling forces of water clusters inside this exclusion region are investigated in this study. With a laser tweezers system, we found the existence of an unexpected force fields inside the solute-free exclusion zone near a Nafion surface. Our results suggest that the water clusters could transduce mechanical signals on the micrometer range within the exclusion zone. This unexpected inhomogeneous force field near the hydrophilic surface would provide a new insight into cellular activities, leading to a potential new physical chemistry mechanism for cell biology.

The effect of atmospheric positivity on the electrical properties of interfacial water was explored. Interfacial, or exclusion zone (EZ) water was created in the standard way, next to a sheet of Nafion placed horizontally at the bottom of a water-filled chamber. Positive atmospheric ions were created from a high voltage source placed above the chamber. Electrical potential distribution in the interfacial water was measured using microelectrodes. We found that beyond a threshold, the positive ions diminished the magnitude of the negative electrical potential in the interfacial water, sometimes even turning it to positive. Additionally, positive ions produced by an air conditioner were observed to generate similar effects; i.e., the electrical potential shifted in the positive direction but returned to negative when the air conditioner stopped blowing. Sometimes, the effect of the positive ions from the air conditioner was strong enough to destroy the structure of interfacial water by turning the potential decidedly positive. Thus, positive air ions can compromise interfacial water negativity and may explain the known negative impact of positive ions on health.

Earlier studies have reported the formation of an exclusion zone devoid of microspheres at the interface of water with a hydrophilic surface such as Nafion® or the hydrophilic ceramic powder. We now report the formation of a ‘three-dimensional cell-like structured exclusion zone’ in water prepared by two different methods. In the first, the hydrophilic powder was agitated with deionized water and allowed to rest (contact method). Subsequently, the ‘powder-supernatant water’ was collected and termed ‘contact water’. In the second method, deionized water in a closed container was kept in the close vicinity of the hydrophilic powder for an extended time-period and it was termed ‘non-contact water’. The two kinds of waters were tested by standard methods for various physical properties. In addition, we carried out cryogenic scanning-electron microscopy of frozen samples of the two kinds of water. The powder-supernatant water showed a cell-like heterogeneous ice structure with the high-density exclusion-zone water forming the walls of a cell-like structure. A similar cell-like ice structure was formed for water treated with the hydrophilic powder in a non-contact manner; the unit cell size depended on the ‘degree of structure’ in the water. When highly structured, the unit cell size was smaller with a concurrently enhanced dielectric constant and reduced redox potential. It was found that the electrical properties are more sensitive to the change in water structure compared to other physical properties such as surface tension, density, and specific heat. Based on our findings of an electric potential difference between the heterogeneous structured water and the ordinary water, we propose a new model to explain the relationship between heterogeneous, structured water and its electrical properties.

Red Blood Cell Behavior within the Exclusion Zone

Simulation and analysis of slip flow of water at hydrophobic silica surfaces of nanometer slit pores