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• Published: 21 July 2022

Water as the often neglected medium at the interface between materials and biology

• B. L. Dargaville   ORCID: orcid.org/0000-0001-5731-0139 1 &
• D. W. Hutmacher   ORCID: orcid.org/0000-0001-5678-2134 1  

Nature Communications volume  13 , Article number:  4222 ( 2022 ) Cite this article

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• Biomaterials
• Small molecules

Despite its apparent simplicity, water behaves in a complex manner and is fundamental in controlling many physical, chemical and biological processes. The molecular mechanisms underlying interaction of water with materials, particularly polymer networks such as hydrogels, have received much attention in the research community. Despite this, a large gulf still exists in applying what is known to rationalize how the molecular organization of water on and within these materials impacts biological processes. In this perspective, we outline the importance of water in biomaterials science as a whole and give indications for future research directions towards emergence of a complete picture of water, materials and biology.

An exceptional molecule

Water is central to all life. Cells, whole organisms and indeed entire ecosystems are fundamentally and completely dependent upon the presence of water. Water is the most abundant substance on earth, making up around 70% of the earth’s surface and 65–90% of the mass of living organisms 1 . Hence, the importance of water in biological processes has been receiving attention in the scientific community for well over a century 2 , 3 .

Water plays an important role in all vital processes of living organisms. All facets of the structure and function of both cells and the extracellular matrix (ECM) are centered around the physical and chemical properties of water. Broad biological functions of water include its action as a transport medium for nutrients and waste products, a medium for chemical reactions, cellular osmoregulation and maintenance of cell turgidity, body temperature regulation, lubrication, pH regulation and the formation of pH buffers.

Water is a complex, structured liquid. It dissolves most biologically important molecules (the notable exceptions being lipids and some amino acids). On the other hand, it is much more than just a passive solvent. Water molecules participate actively as a nucleophile and/or proton donor or acceptor in many chemical reactions in living organisms, such as photosynthesis, cellular respiration, condensation reactions, and hydrolysis of both endogenous and foreign compounds. In addition, over the last several decades it has become apparent that water also plays an active role in many other aspects of the human body and its interaction with foreign substances and surfaces 4 . Much of our understanding of the role of water in biological systems stems from studies of protein and DNA in aqueous solution. Protein-ligand binding, as occurs in the immune response for example, has been suggested to be in part determined by the energetics and dynamics of water 5 . Water—at times individual molecules— facilitates enzyme catalysis and water molecules strongly bound to biomolecules impart thermodynamic stabilization to the latter 5 .

Central to the chemical and physical behavior of water is its nature as a polar molecule. The hydrogen and oxygen atoms have vastly different electronegativities. Thus, the oxygen atom carries a partial negative charge due to its greater attraction for the shared electrons of the H-O covalent bond. Consequently, the two hydrogen atoms carry a partial positive charge. The formation of this dipole results in electrostatic attraction between H and O atoms of adjacent water molecules, generating a type of secondary bonding called hydrogen bonding. Hydrogen bonds are weaker than covalent bonds. The hydrogen-oxygen bond dissociation energies are 21 and 464 kJmol −1 for hydrogen bonds and covalent bonds, respectively 6 . Therefore, within the temperature range for which water is a liquid, hydrogen bonds are able to break and re-form in a continuous dynamic fashion. The lifespan of a hydrogen bond in liquid water is in the range of tens of femtoseconds to picoseconds 7 . Although individual hydrogen bonds are weak, collectively they result in the high cohesive forces of water as a substance 8 , 9 .

The polar nature of water enables crucial cellular functions such as cell membrane formation, support of the three-dimensional shape of the DNA double helix, and it has an important role in the tertiary structure of proteins—specifically, water enables hydrophobic interactions, which are crucial to protein folding and aggregation 10 . Water is a polar, protic solvent and amphoteric reagent, and has the ability to ionize both itself and other molecules. Due to its high heat capacity, water protects against the effects of temperature fluctuation.

There is a vast amount of literature on the behavior of water. The picture is, as yet, far from complete and research in this area is highly active. Entire journal issues have been devoted to the topic of water and its properties 11 . Water is unique among all chemical substances in that it displays many anomalous and unexpected behavior parameters. For example, water has an unusually high boiling point for a substance composed of such small molecules; water displays a decreased viscosity when under pressure; it shows a maximum density at 4 °C and ice has a lower density than liquid water. Other thermodynamic parameters, such as specific heat ( C P ), thermal expansion coefficient (α P ), and compressibility (k T ), all show anomalous behavior 12 . Much of the unusual behavior of water is linked to hydrogen bonding. For example, the high heat capacity and heat of vaporization are due to the large energy input required to break up the hydrogen-bonded network to allow greater molecular movement.

Pettersson et al stated that one of the central questions to the understanding of water is ‘What are the structure and dynamics of the hydrogen bonding network that give rise to its unique properties?’ 11 The introduction of ions and interfaces further complicates the unique properties of water and such scenarios are less well understood than those involving bulk water 11 .

The interaction of water with macromolecules is important on a number of levels, ranging from water associated with both ECM and cellular components such as proteins 13 , 14 to water interacting with drugs, medical devices and implants 15 , 16 , 17 .

The concept of ‘biological water’ has gained prominence in the recent literature. It has been variously defined as any water surrounding a biomolecule; a shell of functional water surrounding a biomolecule; to the notion of cellular water as a distinctive species able to itself perform biological functions 18 . In any event, there is no dispute that a layer of water exists around biomolecules (and other macromolecules), whose properties differ considerably to those of bulk water. In this Perspective Article, we focus on the interaction of water with biomaterials, and more specifically hydrogels, rather than the role of water in the function of endogenous biomolecules.

Hydrogels as the key to understanding the water interface

Water’s exceptional behavior, coupled with its importance in biological systems, has prompted generation of a large body of work with respect to investigation of its properties in the context of biomaterials. Hydrogels are among the most widely used biomaterials for healthcare applications. In addition, hydrogels have been used for applications in other fields such as agriculture, food technology and hygiene. However, biomedical applications represent by far the largest sector, with hydrogels being used widely in areas including pharmaceuticals 19 , diagnostics, tissue engineering and regenerative medicine 20 , drug delivery 21 , 22 , wound dressing, biofiltration, and biosensors 23 , 24 .

A hydrogel is a three-dimensional network of crosslinked hydrophilic polymer chains. The network can swell and hold a large volume of water and has the integrity of a semi-solid material. By definition, a hydrogel contains at least 10 % of its weight or volume as water but may absorb many times its weight in water. The high water content results in mechanical properties similar to natural biological tissue. A distinguishing property of hydrogels is their response to external physical and chemical stimuli, such as temperature, pressure, pH, solvent composition, and the presence of ions and other dissolved species. In this respect and many others, hydrogels have the potential to closely simulate natural biological environments.

Hydrogels can be classified according to their origin (natural, semi-synthetic or synthetic), their ionic charge (cationic, anionic or neutral), or the type of crosslinking involved (covalent, physical, ionic, and others).

Natural hydrogels are derived from polymers such as collagen, gelatin, agarose, alginate, fibrin, chitosan, and hyaluronic acid 25 . Natural hydrogels have been extensively used in numerous biomedical applications, specifically drug delivery and tissue engineering and regenerative medicine research, due to their inherent biocompatibility, biodegradation and bioactivity, such as promoting cell growth 26 . Synthetic hydrogels are based on polymers or copolymers, which include poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), synthetic polypeptides, poly (N-vinyl-2-pyrrolidinone) (PVP), and poly (2-hydroxyethyl methacrylate) (PHEMA). Synthetic hydrogels are, in general, less biocompatible and biofunctional than natural hydrogels; however, they have better mechanical properties and can be easily tailored to different requirements by varying the synthesis parameters.

The overall structure of hydrogels is determined by the type of polymer matrix, degree of crosslinking, porosity and pore structure. However, the central theme of all hydrogels is water. Many of the physical properties of hydrogels are closely related to the water content and organization of water both within the gel and at the gel surface (Fig.  1 ). This organization in turn is dependent upon many factors, both internal (related to the gel composition itself) and external (related to the composition of the surrounding environment) 27 .

a Water content under different swelling conditions. b Comparison of water content in hydrogels with different PEGDA molecular weight. c Comparison of water content in hydrogels with different PEGDA weight fraction. (Figure reproduced from Yang et al, Polymers , 2021, 13 (6), 845, https://doi.org/10.3390/polym13060845 ).

In the 1970s, 80s and 90s a significant amount of work was done on characterization of water states in hydrogels, and a discussion of this body of work appears below. However, with increasing sophistication of hydrogel systems, both natural and synthetic, developed for biomedical use, such fundamental characterization has been largely neglected in recent years.

Many of the early studies were aimed at identifying the presence of different types of water present in polymer systems, initially in natural systems such as proteins and polypeptides, then also synthetic gels such as those composed of PVP and methacrylate polymers. Various designations have been used for the observed water states, including hydration water, associated water, bound versus free water, fast versus slow water, and freezable versus nonfreezable water. One of the first observations was that water within polymers does not display the usual sharp, first-order thermodynamic phase transitions seen for bulk water 28 . Thermodynamic measurements revealed the step-wise nature of the hydration process 29 . A hierarchy was established for the strength of water interactions within proteins: water-ion > water-water = water-polar group > water-hydrophobic group 30 . Clear evidence was compiled for the presence of hydrogen bonding interactions between water and polymer chains in synthetic polymers 31 , 32 , 33 . It was recognized that the behavior of water is sensitive to many different factors, and that different techniques, such as differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR) reveal different aspects of the water phenomena. For example, concentration, temperature, plasticization, polymer mobility and conformation, hysteresis effects, crosslinking and the presence of extra components such as salts, all complicate data interpretation 28 .

The cumulative effort of this era resulted in the ‘three-state’ model of water in hydrogels, wherein water exists in ‘bound’, ‘intermediate’ and ‘free’ states 28 , 34 , 35 . The bound water forms the primary hydration shell around the hydrophilic polymer chains and does not show freezing/melting behavior. Intermediate water forms the secondary hydration shell and exhibits ice melting below 0 °C due to moderately strong interactions with the polymer chains. Free water does not interact with the network structure and behaves very similarly to bulk water, freezing and melting at around 0 °C. The relative content of the three water states depends on many interacting factors, including overall water content. Although only tacitly acknowledged at the time, this creates a fascinating and complex array of possibilities in terms of implications for the biological response to these systems.

Peppas et al carried out work on the thermodynamic interactions of copolymer hydrogels with biological fluids utilizing the Florey–Huggins thermodynamic theory, for the purpose of the evaluation of biomaterials for different applications 36 , 37 . They developed a new method for determining the Florey interaction parameter, χ, for hydrophilic copolymers in contact with water. The work of Peppas, as well as others, allowed prediction of swelling characteristics and solute diffusion for hydrogels designed for biomedical applications, having important implications for protein adsorption, mechanical properties, refractive index, and drug diffusion 38 .

Knowledge of the interaction with water is important from both a biological and a material science perspective: polymer properties depend heavily on the degree and nature of water absorption; while cellular interactions depend both directly on water arrangement at the molecular level and indirectly on the properties of the hydrated polymer.

From a material science point of view, the sorption of water is important for the design of biomaterial properties, understanding plasticization phenomena, and understanding the interaction of polymers with other small molecules 39 . The study of water states sheds light on the pore structure (macroporous, microporous and nanoporous) of hydrogels since water exists in different states in different types of pores and voids 27 .

Over the last decade researchers have synthesized new hydrogels with the emphasis on applications and advanced systems containing cells and other active components for therapeutic use 40 , 41 , 42 . The more fundamental research into molecular structure, water content and structure, and how this relates to functionality has, for the most part, lately been forgotten. Many hydrogels, and biomaterials in general, are lacking the level of function necessary for successful integration into biological systems, and consequently their performance is sub-optimal 43 , 44 . In order to bridge the gap between the emergence of ‘new’ hydrogel systems and successful extension of these to application, a systematic approach is required for the fundamental characterization of the macromolecular interactions in an aqueous environment.

Probing the water phenomena

In order to research water in hydrogels by experimentation, it is a condition sine qua non to build up a fundamental knowledge of the methods involved and their theoretical interpretation, thus assisting in the understanding of the structure and dynamics of bound, intermediate and free water. The study of dynamic processes such as hydrogen bonding and water states necessitates the use of various complimentary experimental techniques. Different methods give varying perspectives on the ‘water state’ phenomenon, since each gives information at different temperatures, different spatial scales and timescales. For example, DSC shows the presence of distinct states of water at low temperatures (in the vicinity of 0 °C) whereas the concept of different states is less applicable at temperatures above ambient (273 K). However, Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA) can give information on water states above 273 K 45 . Another example of this is that NMR and thermally stimulated depolarization current (TSDC) allow study over different temperature ranges (200–280 and 90–270 K, respectively). There has been considerable debate on how to correlate the results from the different techniques 10 .

Methods, such as DSC and NMR, can be used in parallel to gain a deeper insight into hydration and dehydration phenomena of hydrogels. Such combining of techniques allows for a more comprehensive analysis. For example, the parallel use of DSC and X-ray diffraction (XRD) has been used to monitor the growth of ice crystals during the cold crystallization process of intermediate water in the heating cycle of hydrated biomaterials 46 .

Table  1 outlines the experimental techniques for studying water in hydrogels that have advanced the knowledge base, along with some of the materials to which the techniques have been applied. One of the most utilized methods is DSC, in its various forms. It allows quantification of bound, intermediate and free water within gel systems and represents a good starting point for acquiring an overall picture of water behavior in polymeric systems.

Although DSC is extremely functional for quantifying the amounts of the three different states of water, it does not detect water below certain scale threshold levels and cannot be used on its own to construct a picture of events on the molecular and functional group scale. Spectroscopic methods are more likely to reveal this kind of information. 1 H NMR spectroscopy is a more sensitive and much utilized technique. 1 H, 2 H and 17 O T 1 and T 2 relaxation measurements are able to give much of the same information as DSC but over a wider temperature range and can probe a single molecular layer of bound water. 13 C NMR can be used to obtain structural information about the hydrated macromolecules. This can be useful because the network structure and dynamics of the polymer chains may have an important role in regulating the water structure. Yet, direct information about water cannot be obtained from this technique. Infrared spectroscopy allows dynamic probing of the interaction of water with specific functional groups present on the polymer chains but hasn’t thus far allowed quantification of the different types of water present in hydrogels.

Recently, in situ Raman spectroscopy has been employed to identify three distinct types of water, based on differences in their O-H stretching vibrations, in the context of interfacial water involved in electrochemical reactions 9 , 47 . Distinction was made between 4-coordinated, 2-coordinated and ion-coordinated water molecules. Similar methods could in the future be applied to hydrogel systems to probe in detail the exact bonding arrangements that lead to the different states of water observed in these systems.

Several other less common, and therefore to a lesser extent scientifically validated, techniques have been used by a number of research groups to elaborate further on specific aspects of the behavior of bound, intermediate and free water in hydrogels and representatives of these are presented in Table  1 .

Hydrogels are of particular interest with regard to the water-biomaterial account because the development of hydrogels for biomedical applications represents one of the most studied areas at the interface of material science, engineering and medicine. The reason for their attractiveness centers around the ability of these water-rich matrices to mimic natural tissues, both physically and functionally (Fig.  2 ).

a Design of poly- dl -serine (PSer) from l -serine and d -serine. The high l -serine content in silk sericin and the high level of d -serine in the human body as an important neurotransmitter altogether inspired the design of anti-FBR material PSer. b Water solubility of poly-β-homoserine (β-HS) (about 10 mg/mL), poly- l -serine (P- l -Ser) (<0.1 mg/mL due to its β-sheet folding) and PSer (>500 mg/mL). c Circular dichroism spectrum of PSer. d Synthesis of β-HS and PSer. LiHMDS Lithium hexamethyldisilazide, DMAc dimethylacetamide. e Photographs of poly- dl -serine diacrylamide (PSerDA) that was well dissolved at a concentration of 20 wt% and was used to prepare PSer hydrogels by photo-crosslinking in the presence of 0.1% photoinitiator (Irgacure 2959). f PSer hydrogels and PEG hydrogels implanted subcutaneously into C57/BL6 mice induced low FBR and obvious FBR respectively (Figure reproduced from Zhang et al. Nat. Commun. 12 , 5327 (2021), https://doi.org/10.1038/s41467-021-25581-9 ).

The term ‘biocompatible’ is generally used to describe materials which are able to perform a specific biological function while maintaining an appropriate host response. Such materials thus do not give rise to adverse effects when in contact with biological components such as cells, blood and tissues. Specific requirements of biocompatible biomaterials include the ability to resist protein adsorption and cell adhesion, lack of immunogenicity, and being mechanically matched to the host tissue.

The in-depth mechanisms underlying blood and biocompatibility and the overall host response to biomaterials have not been clearly elucidated. However, there have been many attempts in this direction and it appears that the physicochemical properties of hydration water play an important role 48 , 49 , 50 . The blood compatibility of many natural and synthetic polymers has been closely linked to the presence of intermediate water, which appears to prevent direct contact between blood cells, proteins and the material surface 51 . In particular, NMR spectroscopic studies have shown that intermediate water bound to the polymer chains prevents protein adsorption 43 . Other studies have also correlated the presence of hydration water with anti-fouling properties 52 , 53 . Highly hydrated polymers exhibit resistance to non-specific protein adsorption 54 , and changes in hydration due to copolymerization with hydrophobic monomers or increases in temperature 55 , 56 , for example, thus lead to a reduction of non-fouling properties.

Attempts have been made to correlate polymer side-chain mobility, subsequent water mobility and biocompatibility from the viewpoint of protein adsorption 57 , 58 . It was found that the flexibility of both polymer (PMEA>PTHFA>PHEMA) and bound water was directly related to the discovery creating of a marker for biocompatibility, TAT (thrombin-antithrombin III complex) 58 .

As many research groups develop new and more sophisticated hydrogel systems 59 , 60 for biomedical applications, with prominence being given to advanced approaches incorporating cells and other active components, the link between translational and fundamental research into the role played by different water states in material end-function is more important than ever.

As described in the preceding sections, the molecular mechanisms underlying the interactions of water molecules with biomaterials, specifically polymer networks such as hydrogels, have been well-studied and although there is much still to be learned, progress has been achieved in understanding these processes. However, a significant gap still exists when it comes to applying this knowledge to rationalize the way in which the molecular organization of water on the surface and within biomaterials affects their in vitro and/or in vivo biocompatibility.

Biotherapeutic environments are complex and consist not only of tissue, implant and water, but also a multitude of other molecules and species in the cellular and extracellular space. Another layer of the puzzle that warrants consideration concerns the presence of these species (which include metabolites, electrolytes and other osmolytes) and their influence on the interaction of water with hydrogel macromolecules and the subsequent biological response. While it has been anticipated that they will affect the properties of water, exactly how this occurs has been controversial and there is a distinct lack of research considering this problem. Most researchers traditionally frame their experimental models against a pure water setting (Fig.  3 ). Clearly, the biological relevance of such an approach is limited.

Inset: The same data presented on a logarithmic scale to enable easier visualization. Source of data: Web of Science. See  Supplementary Information for details of the method used to conduct the search.

Figure  3 displays the number of papers in the biomedical literature, published since 2000, that have studied hydrogel swelling, compared to the number of those same papers that use PBS as the swelling medium. ‘PBS’ was chosen as a search term in order to represent those studies that have considered more physiologically relevant swelling media than pure water. Although it can be conceded that this data excludes a number of studies which utilize other more complex osmotic and biologically relevant fluids, the number of such studies is very small compared to those that use PBS, and their omission does not alter the overall trend shown. Indeed, a closer perusal of the studies that do not involve PBS reveals that most of them do in fact use pure water as the swelling medium.

The rapidly emerging design space of hydrogels with controlled physical, chemical and biological properties represent a great opportunity for future research. We propose that future strategies in this area should involve pairing what is known from the early fundamental research on water in hydrogels with the more recent work on the role of water in biological systems and applying this to the characterization of newly developed hydrogel systems. Detailed studies in this direction will not only enable filling of some of the knowledge gaps that exist hitherto, but will also enable the development of a more systematic approach for hydrogel characterization. Only in this way will a more complete scientific framework of biocompatibility, water and materials emerge. This may indeed require the development of new experimental and theoretical techniques to further probe the dynamics of this topic and answer the many questions that remain regarding the big picture of water in biomaterial science as a whole.

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A Novel Tool for Visualization of Water Molecular Structure and Its Changes, Expressed on the Scale of Temperature Influence

Zoltan kovacs.

1 Department of Physics and Control, Faculty of Food Science, Szent István University, H-1118 Budapest, Hungary

Bernhard Pollner

2 Department for Hygiene and Medical Microbiology, Medical University of Innsbruck, A-6020 Innsbruck, Austria; [email protected]

George Bazar

3 Department of Nutritional Science and Production Technology, Faculty of Agricultural and Environmental Sciences, Kaposvar University, H-7400 Kaposvar, Hungary; uh.balirga@razab

Jelena Muncan

4 Biomeasurement Technology Laboratory, Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan; pj.ca.u-ebok.elpoep@nacnumj

Roumiana Tsenkova

Aquaphotomics utilizes water-light interaction for in-depth exploration of water, its structure and role in aqueous and biologic systems. The aquagram, a major analytical tool of aquaphotomics, allows comparison of water molecular structures of different samples by comparing their respective absorbance spectral patterns. Temperature is the strongest perturbation of water changing almost all water species. To better interpret and understand spectral patterns, the objective of this work was to develop a novel, temperature-scaled aquagram that provides standardized information about changes in water molecular structure caused by solutes, with its effects translated to those which would have been caused by respective temperature changes. NIR spectra of Milli-Q water in the temperature range of 20–70 °C and aqueous solutions of potassium chloride in concentration range of 1 to 1000 mM were recorded to demonstrate the applicability of the proposed novel tool. The obtained results presented the influence of salt on the water molecular structure expressed as the equivalent effect of temperature in degrees of Celsius. The temperature-based aquagrams showed the well-known structure breaking and structure making effects of salts on water spectral pattern, for the first time presented in the terms of temperature influence on pure water. This new method enables comparison of spectral patterns providing a universal tool for evaluation of various bio-aqueous systems which can provide better insight into the system’s functionality.

1. Introduction

Recently, water has become more and more important subject of studies, as evidenced by the increasing number of water-related publications in various fields of science [ 1 , 2 , 3 , 4 ]. Water is fundamental to life—it is one of the essential and widely distributed components of biologic systems, which can be considered as a biomolecule in its own right [ 5 , 6 ]. Different scientific fields have been studying water from different aspects, trying to reach better understanding of its properties, structure and functions, yet still, our picture of water as a substance is rather incomplete. Spectroscopy methods, which are based on interaction of light and matter, have contributed a lot to our understanding of water. Recently, established, scientific discipline of aquaphotomics aims to integrate the knowledge these methods acquired about water based on its interaction with light, into one “omics” discipline which relates the structure of water with its functionality, and whose ultimate objective is better understanding of water as a matrix of aqueous and biologic systems [ 7 ]. In this regard, aquaphotomics have made significant novel discoveries, and utilized the properties of water in various application fields ranging from water and food quality monitoring, microbiology, to biomeasurements, biodiagnostics and biomonitoring [ 8 , 9 , 10 ]. Owing to the wide range of aqueous systems and biologic systems aquaphotomics studied, some novel, surprising insights have been discovered, which place focus on importance of water structure, hydrogen bonding and temperature.

The effect of temperature has always been one of the most studied phenomena in spectroscopy with respect to its influence on water structure. The very terms “structure-makers” and “structure-breakers” are coined to describe substances which induce changes in the water structure and consequently its spectrum, comparable to decrease in temperature and increase of temperature, respectively. Effects of salts or sugars are commonly described using those terms [ 11 , 12 , 13 ]. The known influence of temperature on hydrogen bonding in water is something that can be used as an etalon for better understanding of how substances affect the structure of water in various solutions. Among many others, principal component analysis and two-dimensional correlation spectroscopy [ 14 ] or multivariate curve resolution-alternating least squares technique [ 15 ] were used to describe the effects of temperature perturbations on the NIR spectra of water in terms of hydrogen bonding either alone or in comparison to salt perturbation.

Aquaphotomics studies made some further steps uncovering that various phenomena, related to biomolecules or functionality of living organisms and aqueous systems can be described as related to specific water molecular structure and presented using spectral pattern.

For example, in one work, concerned with the detection of UV-induced damage on DNA structure, it was found that the aqueous solutions of UVC-damaged DNA caused increase in hydrogen bonded water—i.e., that damaged DNA was a structure-making element causing changes of water similar to low temperature [ 16 ]. In another study, which explored cold tolerance ability of different soybean cultivars, it was found that the water structure in the leaves of those cultivars with higher cold tolerance ability even at low temperatures preserved the water in less-hydrogen bonded state compared to those cultivars who are more susceptible, as if the environmental temperature would have been in fact higher [ 17 ]. What these novel insights into the water structure and analogy with temperature influence provided is a novel knowledge and better understanding of the water functionality at different levels of organization of biologic systems.

One of the main visualization and analytical tools of aquaphotomics, is the so-called aquagram [ 8 , 18 ] which provides a comprehensible demonstration of the ratios of different water species present in a sample. Aquagrams have been found very useful in many applications, such as diagnosis of estrus in giant pandas [ 19 ], orangutans [ 20 ] and cows [ 21 ] and showing the different water spectral patterns of probiotic and non-probiotic bacteria strains [ 22 ], or for example, revealing different types of water in the soft contact lenses, in a completely nondestructive manner [ 23 , 24 , 25 ]. The cited references demonstrated the usefulness of the presentation of the water spectral patterns in aquagrams.

In order to unify the relation between water species depicted by aquagrams and related water functionalities we propose to use temperature as a common denominator to express changes of light absorbance at each of the water vibrational frequencies as changes caused by the most influential perturbation for water—temperature. Considering the advantages of better understanding of the functionality of water structure if the analogy is made with the influence of temperature, the objective of this work was to develop a novel method and a visualization tool, so called temperature-based aquagram which translates the effects of any type of perturbation of water structure in aqueous or biologic systems to the equivalent effects that would have been caused by the temperature changes and expressed in temperature units. The need to introduce the temperature-based aquagrams arose from the experiments on more complex systems, such as previously described, where it was observed that certain phenomena, (caused by solutes for example) have effects on the water molecular structure of the system and contribute to its functionality in the way analog to the changes in temperature.

In a study concerned with classification of bacteria based on the probiotic strength it was found that the strong probiotic bacteria, compared to the moderately strong strains or non-probiotic strains, create less hydrogen-bonded water species ( Figure 1 ) [ 22 ]. On the other hand, the spectra of pure water at different temperatures show shift of the main band towards the shorter wavelengths with increasing temperature [ 14 ], i.e., showing that increase in temperature results in breaking of the hydrogen bonds. If the two cases are compared, it can be seen that probiotic bacteria affect surrounding water similarly to the influence of temperature increase, as if they are a structure-breaking element [ 22 ]. This conclusion supports the novel insight into molecular mechanisms of how probiotics work–they increase solubility of substances in water [ 26 ], just like the increase in temperature of the water would contribute to better solubilization.

Water spectral pattern presented on aquagrams shows different water structure in different bacteria strains (Reprinted with permission from Slavchev, A., Kovacs, Z., Koshiba, H., Nagai, A., Bázár, G., Krastanov, A., Kubota, Y. and Tsenkova, R. 2015. [ 22 ]).

From this example, it can be seen that comparisons with effect of temperature can be useful and contribute to better intuitive understanding of the functionality of the studied system. Recalling the soybean cultivars example, the role of many substances that plants accumulate in response to cold stress, becomes more obvious—their function is to provide a certain water molecular structure in the leaves as if the temperature of the environment is different [ 17 , 27 ].

The concept of the novel visualization tool—a temperature-based aquagram—was developed similarly to the one Bernal and Fowler introduced long ago—the so called “structural temperature” concept [ 28 ]. The “structural temperature” is the respective temperature at which pure water would have effectively the same molecular structure as the water of the aqueous system under study. At the time of introduction, no adequate method that could describe that structure was found; there were propositions that it can be estimated based on the measurements of viscosity, Raman spectra or X-ray diffraction. In addition, the intended purpose was mainly concerned with the applications in analysis of electrolyte solutions, and since the concept did not offer the possibility to separate effects of the individual ions, the entire idea was more or less abandoned [ 29 ]. However, the structural temperature concept fits well into the framework of aquaphotomics, which places the water spectral pattern and the respective water functionality of the system in the central place. In contrast to “reductionistic omics methods” which are focused on isolating the biomolecules and separation of elements of the system, the aquaphotomics views each aqueous or biologic system as a whole, where all the components of the system exert their influence on the water matrix, whose structure is directly related to the function of the system [ 9 , 10 ]. Just like the temperature is the macroscopic, measurable characteristic, arising from the molecular structure of the system, so is the light absorbance at each water specific vibrational frequency, and one can benefit from expressing one in the terms of another.

The purpose of this study is to introduce a novel visualization tool, which expresses the effects of any type of perturbation of water molecular structure in aqueous or biologic systems to the equivalent effects that would have been caused by the temperature and expressed in temperature units. To illustrate this, we have chosen a simple salt solution and a concentration as a major perturbation. Salt is chosen as it is not near infrared active substance, hence the changes in absorbance of the solutions are only due to the changes in water molecular structure [ 15 , 30 ]. Following the steps provided in the study, one can easily replicate the experiment, develop the temperature-based aquagrams and use it further for specific purposes. While in this study, the analysis and the results are presented for only 1st overtone of water region, the same methodology is applicable for any region of the water absorbance spectra. This new method provides numerical results on a clearly defined scale with confidence intervals. It enables the comparison of results across time and different experiments and provides information about the statistical significance of the found differences.

2. Results and Discussion

The spectral data in the wavelength interval of 1300 to 1600 nm for the two experiments were separately subjected to principal component analysis (PCA). The PCA score plots demonstrated the multidimensional patterns of the spectral data. Specifically, the highest variations displayed in the first principal components (PC1) were related to temperature and concentration of potassium chloride, representing 99.3% and 99.1% of the spectral variation in case of the temperature and the potassium chloride experiments, respectively. The PCA results did not show outliers either in the temperature or the potassium chloride datasets. The raw and 2nd derivative spectra of the two experiments were analyzed separately to discover the wavelengths exhibiting the largest changes caused by the temperature and salt perturbations.

2.1. Results of Temperature Experiment

The raw and 2nd derivative absorbance (logT −1 ) spectra in the spectral range of 1300 to 1600 nm of Milli-Q water in the temperature range of 20 to 70 °C are shown in Figure 2 . The main feature of the NIR spectrum of water is a broad peak around 1450 nm, comprised of several overlapping bands, described mainly as the 1st overtone of the OH stretching vibration [ 31 ]. This observation is confirmed by the second derivative spectra which indicated very intense bands at 1412 and 1462 nm. These bands are well known as bands associated with free water molecules [ 32 , 33 , 34 ] and strongly hydrogen-bonded water [ 14 , 35 ], respectively.

Raw and 2nd derivative (calculated with Savitzky–Golay filter using 2nd order polynomial and 21 points) absorbance (logT −1 ) spectra in the spectral range of 1300–1600 nm (OH first overtone) of Milli-Q water in the temperature range of 20–70 °C ( n = 78).

From a spectroscopic point of view, an increase in temperature has been interpreted as a decrease of number of hydrogen bonds [ 14 , 34 ], while others explain it via weakening of hydrogen bonds [ 36 ]. There is agreement, regardless of one’s preferred theory, that the change of temperature causes alteration in hydrogen-bonding configurations of water as described in more details previously [ 37 ].

Figure 2 shows a “blue shift”, i.e., movement of the main band towards the shorter wavelengths with increasing temperature and an isosbestic (temperature invariant) point. These phenomena are well studied in scientific literature [ 14 , 34 , 38 ]. Evidence clearly suggest that less hydrogen-bonded water is predominant at higher temperature, while spectra acquired at low temperature are represented mostly in the more hydrogen-bonded area where water molecules form species with one (S1), two (S2), three (S3) and four (S4) hydrogen bonds [ 39 ].

Despite the competing theories about the water structure, aquaphotomics identifies water-specific absorbance bands with higher variations caused by respective perturbation for the system of interest and uses them to depict the unique spectral pattern as an integrated marker of the system–perturbation interaction. The presentation of the 12 specific water coordinates [ 7 ] of the spectra of Milli-Q water acquired in the temperature range of 20 to 70 °C together with the 95% confidence intervals is given in Figure 3 a,b, calculated with the temperature-based aquagram calculation method. In the case of working with more complex system, it would be advisable to follow the protocol of aquaphotomics analysis which can provide more thorough examination of activated water absorbance bands and not necessarily limit the presentation to only the 12 coordinates as is chosen here. The aquagram provides an easy-to-comprehend presentation of the phenomena described above, i.e., the change of the strongly and weakly hydrogen-bonded patterns, based on Figure 2 . The movement of the higher absorbance towards shorter wavelengths with increasing temperature is convincing. The scales of the aquagrams ( Figure 3 a,b) express the effect of perturbation occurring at the 12 coordinates (i.e., in the defined wavelength ranges) in degrees Celsius equivalent. Therefore, in this specific example the radial values show the temperatures corresponding to the signal acquisitions (20 to 70 °C). The plotted dashed and dotted lines represent the upper and lower confidence levels of the aquagram values, respectively, for each single analyzed temperature step. There was no overlapping of the confidence intervals (95%) of the Milli-Q samples measured at different temperature observed, meaning that 2 °C temperature changes caused statistically significant effects on the individual coordinates (i.e., in the defined wavelength ranges). The stability of the temperature-based aquagram calculation is presented in Figure 3 b, by depicting only three selected temperature levels (20, 30 and 40 °C). Though the spectral dataset used to calculate the temperature-based aquagram of Figure 3 b is only a subset of the dataset used to calculate Figure 3 a, the shape and the range are the same in both cases. This type of stability was not possible with the “classic” aquagram ( Figure 3 c,d). These examples present the applicability and the additional benefits of the newly developed temperature-based aquagram for the evaluation and presentation of the water species in the 12 coordinates (representing defined wavelength ranges) through the evaluation of a well-known perturbation, i.e., the effect of temperature on the water spectral changes.

Aquagrams of Milli-Q water in the temperature range of 20–70 °C, ( a , b ) with 95% confidence intervals calculated with temperature-based aquagram calculation method, ( c , d ) calculated with the classic calculation method, ( a , c ) all the 26 temperature steps ( n = 78) and ( b , d ) on three selected temperature steps ( n = 9) to show the stability of the methods (UCL—upper confidence level, LCL—lower confidence level).

2.2. Results of Potassium Chloride Experiment

The raw and 2nd derivative absorbance (logT −1 ) spectra of the aqueous solutions of 0.001 to 1 M potassium chloride salt in the 1300–1600 nm wavelength range (OH first overtone) are presented in Figure 4 . The main component of the aqueous solution, other than water, is KCl, which has no absorption in the NIR region; thus, it is not surprising that the spectra show a broad peak around 1450 nm. The second derivative spectra also provide similar patterns to those of the Milli-Q water acquired at different temperature, indicating bands at 1412 and 1462 nm. The trend of the shift in the peak position was similar to that observed in the temperature-perturbed pure water (i.e., the peak moves towards lower wavelengths as temperature is increased), but the actual peak locations were different from those for the pure water. The effect of low concentrations of salts diluted in water has been illustrated by the changes in the OH bonding of water molecular systems in many experiments [ 15 , 40 , 41 , 42 ]. As salts do not absorb NIR light, accurate measurement of even low concentration of salts, published in the above-mentioned papers, means salts change the surrounding water molecular structure according to the number of the solvent molecules in the solution which still can restructure the water molecular system.

Raw and 2nd derivative (calculated with Savitzky–Golay filter using 2nd order polynomial and 21 points) absorbance (logT −1 ) spectra in the range of 1300–1600 nm (OH first overtone) of 0.001–1 M KCl solutions ( n = 180).

This phenomena is not new; Bernal and Fowler [ 28 ] showed that the addition of electrolytes changes the spectrum of water in the infrared overtone region. Lin and Brown [ 43 ] also analyzed the effects of different salts on water spectra, and the authors were able to build accurate regression models using the spectral range of 1490 to 1610 nm for salt content prediction.

The spectra shown in Figure 4 imply that the increasing concentration of KCl causes “blue shift”, i.e., a shift towards the spectral range referring to the band of less hydrogen-bonded water molecules. Our findings, hence show a good agreement with the results of previous research [ 15 , 40 , 41 , 42 ].

A more detailed evaluation of the proposed 12 specific water coordinates [ 7 ] of the spectra of the aqueous solutions of potassium chloride compared to the spectra of water is provided in Figure 5 . The plots show the aquagrams of the single concentration levels together with the respective 95% confidence intervals calculated with the “classic” method ( Figure 5 a) and the new temperature-based aquagram calculation method ( Figure 5 b). The relative position of the patterns of the single concentration levels shows some similarity for the two methods. Furthermore, in both methods, spectral patterns of the highest concentration range (100–1000 mM) show the highest difference from the spectra of the lower ranges and Milli-Q samples (the latter one in black). This pattern explains the same phenomena which were found based on the raw spectra, i.e., a higher concentration of salt create water with less hydrogen bonds and decreases the number of water species to more hydrogen bonds. For the higher concentration ranges the aquagrams of the individual concentrations do not overlap, i.e., addition of 0.01 or 0.1 M KCl caused statistically significant change at the individual coordinates.

Aquagrams with 95% confidence intervals of Milli-Q water ( n = 150) and 0.001–1 M KCl solutions ( n = 180) calculated with the classic ( a ) or the temperature based aquagram ( b ) calculation methods on the individual concentrations (UCL—upper confidence level, LCL—lower confidence level).

However, the results also show an increasing tendency of the C7 coordinate (1432–1444 nm) with increasing concentration of KCl, which can be assigned to water molecules with one hydrogen bond (S1), meaning that increase in concentration of salt leads to increase in the number of these molecules [ 39 ].

The dominantly higher effect of the higher concentrations on the spectral pattern hides the pattern of the lower concentrations; therefore, the calculation of the aquagrams were performed using the spectral data set for the two lowest concentration ranges only ( Figure 6 ). The phenomena of the higher concentrations on the water spectral pattern, namely the higher concentration being more dominantly a structure breaker, can be observed in case of the concentration of samples at the concentration range of 10 to 100 mM. It is interesting to note the similarities to the pattern of the aquagrams calculated on all the three ranges ( Figure 5 b).

Aquagrams with 95% confidence intervals of Milli-Q water ( n = 150) and 0.001–0.1 M KCl solutions ( n = 120) calculated with the temperature based aquagram calculation method on the individual concentrations (UCL—upper confidence level, LCL—lower confidence level).

The aquagrams of the higher concentrations (10–100 mM) show consistency with the previous findings regarding the structure-breaking characteristic as displayed in Figure 6 . More specifically, the higher concentration samples present higher absorbance values in the range between 1342 and 1374 nm, i.e., C01-C03 that refer to the 1st overtone of free OH stretch (OH–(H 2 O) n , n = 1…4) [ 44 , 45 ] and 1440 and 1452 nm, i.e., C07-C08 that are known as the bands of water hydration [ 35 ] and water molecule connected to another water molecule (S 1 ) [ 14 , 46 ] and the symmetric and asymmetric stretching of first overtone of water [ 35 , 46 , 47 ]. However, in the range between 1476 and 1512 nm, i.e., C10-C12, the higher concentration samples show lower values and these wavelengths are usually assigned to strongly hydrogen bonded water [ 14 , 35 ] and aqueous protons ([H + •(H 2 O) 6 ]–H 2 O in H 5 O 2 + symmetric stretch) [ 48 ]. These findings also mean decreasing concentration of salt causes a shift towards longer wavelengths, i.e., the range referring to the band of more hydrogen bonded water molecules.

Using the additional benefit of the temperature-based aquagram calculation method, further results can be achieved from Figure 6 . The addition of, for instance, 0.1 M potassium chloride to Milli-Q water results in water structural changes equivalent to changes caused by temperature of about 0.65, 0.6, 0.3, 0.1, 0.2 0.6, 1.8, 1.2, 0.2, −0.1, −0.3 and −0.6 °C at C01, C02, C03, C04, C05, C06, C07, C08, C09, C10, C11 and C12 coordinates, respectively. Furthermore, having calculated the confidence intervals, the statistical significance of the differences is also available. For example, calculations showed that addition of 0.02 M KCl to Milli-Q caused change equivalent to the change due to 0.33 and 0.23 °C temperature increase at coordinates C07 and C08 when compared to pure Milli-Q, which was found significant ( p = 0.05), too.

The aquagrams of the lowest concentration range (1–10 mM) calculated with the temperature-based aquagram method is shown in Figure 7 to further evaluate the findings of the effects of decreasing concentrations of salt. The results suggest that we can observe concentration levels of 5 to 10 mM that cause a shift towards longer wavelengths, i.e., the range referring to the band of more hydrogen-bonded water molecules compared to the spectrum of Milli-Q water. These findings imply that salts can also have structure-breaker and structure-maker effects on water structure similar to the behavior of sugars [ 13 ]. However, the aquagrams of the even lower concentrations (1 to 5 mM) also show alteration with nearly the same deviation, but towards shorter wavelengths. This may suggest that changing between structure-breaker and structure-maker properties of salt exist at such a low concentration, but the discernment of these minor changes would require very accurate measurements.

Aquagrams with 95% confidence intervals of Milli-Q water ( n = 150) and 0.001–0.01 M KCl solutions ( n = 60) calculated with the temperature based aquagram calculation method on the individual concentrations (UCL—upper confidence level, LCL—lower confidence level).

3. Materials and Methods

3.1. samples.

Two experiments with different sources of perturbations were conducted to demonstrate the procedure of temperature-based aquagram development and show the advantages in comparison to the representation of spectral data using classic aquagrams. In both experiments, pure Milli-Q water was used as a sample (Milli-Q purification system (Millipore, Molsheim, France, resistance = 18 MΩ) and in the first experiment perturbation of the sample was caused by changes in temperature, while in the second one, by changes in the concentration of salt.

3.2. The Temperature Experiment

The effect of temperature perturbation on water near-infrared spectra has been well-studied and is thoroughly described in the literature [ 14 , 34 , 49 ]. Therefore, the experiment was performed on Milli-Q water in the temperature range of 20 to 70 °C to acquire spectra which could be used for the evaluation of the aquagram methods. The Milli-Q water was produced by a Milli-Q purification system (Millipore, Molsheim, France, resistance = 18 MΩ). The spectral acquisition was performed at 2 °C increments, resulting in 26 temperature steps in the range of 20 to 70 °C.

3.3. The Potassium Chloride Experiment

The addition of salt to water at different concentrations is also an often evaluated perturbation [ 42 , 50 ]. Therefore, an experiment was performed with different concentrations of aqueous solutions of potassium chloride. Potassium chloride (KCl, M = 74.56 g mol −1 , purity min. 99.0% mass/mass) was purchased from Wako Pure Chemical Industries, Ltd. (Kobe, Japan). Aqueous solutions were prepared in different concentrations of KCl, in the range of 1 to 1000 mM. Three concentration ranges were prepared: Range A, from 100 to 1000 mM concentration, in steps of 100 mM; Range B from 10 to 100 mM, in steps of 10 mM and finally, Range C, in 1 to 10 mM in 1 mM concentration steps. Each dilution was prepared by serial dilution from the stock samples with the highest concentration in a given range (A, B or C) and prepared in two replicates, resulting in two independently prepared sets of samples. Stock solutions were prepared and further serially diluted with added Milli-Q water step-by-step to reach the appropriate concentrations—a solution created in each step was further diluted to prepare the next lower concentration.

3.4. NIR Spectral Acquisition

A FOSS-XDS spectrometer (FOSS NIRSystems, Inc., Hoganas, Sweden) equipped with a Rapid Liquid Analyzer module including a temperature-controlled 1 mm pathlength cuvette holder was used to measure transmittance spectra (logT −1 ) of the Milli-Q samples for the temperature experiment and of the aqueous solutions for the KCl experiment. Spectral acquisition was performed by saving three consecutive spectra in the range of 400–2500 nm at 0.5 nm spectral steps. Each saved spectrum was the average of 32 successive scans.

A thermal bath with continuous water circulation was attached to the Rapid Liquid Analyzer module to ensure the required temperature of the sample during scanning in the range of 20 to 70 °C at 2 °C increments.

The same apparatus was used to provide a constant temperature of 28 °C where each aqueous solution of potassium chloride was incubated for 90 s to equilibrate to the required temperature before scanning. Milli-Q water samples were measured as every fifth sample during the KCl experiment to provide environmental controls.

The total number of spectra for the temperature experiment was 78 (26 temperature steps × 3 consecutive scans) and for the KCl experiment was 330 (30 concentrations × 2 repeats × 3 consecutive scans + 150 Milli-Q control scans) ( Appendix A Dataset).

The FOSS-XDS instrument was operated using VISION 3.5 software (FOSS NIRSystems, Inc., Hoganas, Sweden). In both experiments, reference spectra were recorded before every sample.

3.5. Statistical Data Analysis

Only the wavelength interval of 1300 to 1600 nm, corresponding to the first overtone of the O–H stretching band [ 51 ] was used for the evaluations. For the purpose of explaining methodology of how to develop temperature-based aquagrams, in this study, the focus is placed on this particular part of the water absorbance spectra, because it is best understood so far in the terms of the water molecular species whose absorbance bands are well-resolved and their assignments known [ 7 ]. In the first overtone of water as a result of systematization of experimental work done on many different systems, not only different aqueous solutions, but also a great variety of biologic systems under different perturbations 12 water absorbance bands termed WAMACs (Water Matrix Coordinates)–each range from 6 to 12 nm width, were discovered [ 7 ]. The great body of evidences in scientific literature provided the meaning to these ranges—i.e., it was possible to connect each of these 12 coordinates to specific water molecular species. In aquaphotomics studies the 12 WAMACs are called, coordinates because they represent windows in the spectra through which water structure of the system under study can be observed. In this study, the 12 WAMACs will be used to develop aquagrams, but it should be noted that the methodology explained is applicable for any region of the water absorbance spectra; it is not necessarily limited to the coordinates chosen here. Here, we chose for the reason of simplicity and because they are well-understood to use only those. However, depending on the system under study and the range of the spectra used, the reader is advised to follow the aquaphotomics protocol of analysis for extraction of the “activated water absorbance bands–WAMACs [ 8 ] and then follow the further instructions to develop temperature-based aquagrams as described below.

Before the development of aquagrams, exploratory analysis was performed. First, Principal component analysis (PCA) [ 52 ] was used to describe multidimensional patterns in the spectral data and to discover outliers. The raw and 2nd derivative spectra were plotted to visualize the spectral changes induced by temperature perturbation and by the perturbation of salt concentration on the spectra of Milli-Q water and aqueous solutions of potassium chloride, respectively. The 2nd derivative spectra were calculated using a Savitzky–Golay filter [ 53 ] using the 2nd order polynomial and 21 points.

3.5.1. Calculation Protocol of “Classic” Aquagram

The absorbance values at specific water matrix coordinates (WAMACs) [ 7 ] define the water spectral pattern (WASP), which is different for different perturbations. The WASP can be visualized by a chart called the aquagram [ 18 , 19 ]. This representation of the WAMACs was first introduced by Tsenkova [ 18 ]. This aquagram (from now on called the “classic” aquagram) displays the multiplicative-scatter-corrected (MSC) (or standard-normal-variate (SNV)) transformed, normalized and averaged absorbance values of different samples or sample groups at 12 specific characteristic wavelengths. As, mentioned, these specific wavelengths were experimentally discovered as absorbance bands of specific water molecular species in previous studies and are later confirmed by overtone calculations of already reported water absorbance bands in the infrared range [ 7 ]. The aforementioned water absorbance bands cover various form of water molecular species and are thus useful to depict characteristic spectral patterns in the first overtone region of water. This calculation can be summarized by Equation (1).

where, A λ ′ —value on aquagram for a given wavelength; A λ —absorbance after MSC applied on 1st overtone region of OH (i.e., 1300–1600 nm); μ λ —mean of all spectra for the examined group at a given wavelength (after MSC applied); σ λ —SD of all spectra for the examined group at a given wavelength (after MSC applied); λ —12 wavelengths (1342, 1364, 1374, 1384, 1412, 1426, 1440, 1452, 1462, 1476, 1488, 1512 nm) [ 7 ].

The classic aquagram shows the relative fingerprint, i.e., the WASPs, in the context of all spectra in the examined group [ 18 , 19 , 21 , 54 ].

Recently, this aquagram calculation method was extended by adding the possibility to observe the statistical significance of the differences presented on the aquagrams. Therefore, besides the average spectra of the individual groups used to plot the aquagrams, the respective confidence intervals are also calculated using the so-called Bootstrap method [ 55 ]. This improvement makes it possible to plot the aquagrams together with their upper and lower 95% confidence interval limits.

3.5.2. Calculation Protocol for Newly Developed (Temperature-Based) Aquagram

The newly developed temperature-based aquagram calculation algorithm presents the respective water matrix coordinates in units equivalent to change in temperature and includes the respective confidence intervals. Therefore, it gives rise to the possibility to compare the WAMACs across time and also across different experiments and it provides information about the statistical significance of the differences.

The new calculation method is based on the comparison of the areas (under the ranges of the above-mentioned 12 specific water coordinates [ 7 ]) of the respective test sample spectra and the spectra of Milli-Q water. This method aims to express the spectral pattern changes in units equivalent to the change of temperature that would cause the observed change. The calculation of this aquagram concept (from now called the temperature-based aquagram) can be summarized in the following steps. Note that the calculation steps are explained for one coordinate (C01 representing spectral range between 1336 and 1348 nm, as an example) out of the 12 coordinates (C01-C12, i.e., defined wavelength ranges) to give an easily understandable description, but the same steps have to be repeated for each of the 12 coordinates. The main calculation steps are summarized in a chart to provide an overview of the developed method ( Appendix B Figure A1 ).

The dataset of the experiment of interest is defined as the experimental dataset.

The average spectra of the groups of interest (in this case, salt concentration levels) are calculated in the experimental dataset together with their respective confidence intervals using the Bootstrap method [ 55 ], yielding as many single, unique spectra as there are groups are in the experimental dataset (plus their upper and lower 95% confidence interval limits).

Scheme of the calculation for Area under the curve (AUC) aquagram method. Spectrum of pure water with highlighted subranges of the 12 specific water matrix coordinates (WAMACs).

• Step 4. The ratio of the area under the curve for each single coordinate is calculated with respect to the full area under the curve for the first overtone OH region (i.e., the area of C01 is divided by the full area under the spectrum in the range of 1300 to 1600 nm). This is done for every single average spectrum, in the reference dataset and the experimental dataset (together with the respective confidence interval limits for the experimental dataset). This calculation step provides normalized values and avoids possible differences due to scattering and/or pathlength effects.
• Step 5. Based on the reference dataset, a continuous array of values for the relative area of C01 (as calculated in Step 1) is calculated for a continuous temperature range from 20 to 70 °C using local polynomial regression. This is an essential step in order to accommodate the data from an experiment performed at specific temperature—see Step 6.
• Step 6. The basic principle of the temperature-based aquagram method is to compare the effect of the perturbation used on the system under study which resulted in a certain water spectral pattern to the effect the temperature changes would induce in pure water. Thus, any perturbation can be expressed as an equivalent temperature effect on a Milli-Q water sample. It is necessary to perform a “local calibration” with the reference dataset around the temperature of the experimental dataset. Therefore, in this step, the temperature calibration range is defined. This range is used to express the effect of perturbation in degrees Celsius equivalent. For this, a symmetrical scale is defined from the reference dataset (calculated at Step 5) using two degrees, plus and minus around the temperature of the experiment (hence, a span of 4 °C). For example, if the experiment was performed at 25.0 °C, then the calibration range of 23.0 to 27.0 °C would be used.
• Step 7. The temperature calibration equation, the relationship between the change of the temperature and change of the area of C01 at the temperature of the experiment, is determined based on the calculation performed in Step 5 on the reference dataset. (It is known how the area of C01 changes as a function of temperature described by a linear function). Therefore, it is easy to compare the changes for areas for C01 for the experimental dataset (calculated at Step 3) to the changes of the area of C01 caused by temperature, i.e., to express the changes in C01 in units of temperature (degrees Celsius) equivalent.
• Step 8. The calculated temperature (degrees Celsius) equivalent value for every group of the experimental dataset is finally visualized together with the respective 95% confidence intervals in a radar chart, where the units of the axes are in degrees Celsius.
• Step 9. The calculation and visualization of the results were performed using the R programing language [ 56 , 57 ].

4. Conclusions

Recently, aquaphotomics has been introduced to focus on water as a key component for providing information about the function of the entire system. Non-destructive NIR spectroscopy and aquaphotomics have been applied to explain new phenomena in the field of life sciences. In contrast to reductionistic methods in which biomolecules and other elements are analyzed separately from the system, aquaphotomics studies the aqueous systems through its water matrix.

In the present study, a newly developed temperature-based aquagram calculation method is presented as an additional tool to express the changes of water molecular structure in aqueous systems which are caused by perturbations different from temperature. Although the need to introduce temperature-based aquagrams originated from experiments on complex systems, the successful application of the new aquagram calculation method was demonstrated through the evaluation of the results of well-known phenomena. The results of temperature and salt perturbations on Milli-Q water are demonstrated in the present study.

The effect of temperature on the spectral pattern of Milli-Q water acquired in the temperature range of 20 to 70 °C is presented with the temperature-based aquagram calculation method. The method provides the presentation of the phenomena demonstrating blue shift in the first OH overtone range with increasing temperature. Furthermore, the scale of the aquagram expressed the effect of perturbation in degrees Celsius equivalent.

Aqueous solutions of potassium chloride were chosen for the experiment as KCl has no absorption in the NIR region. Thus, its effect on water spectral patterns can be clearly evaluated and presented as caused by temperature changes at respective wavelengths. Furthermore, it demonstrates that the method is invariably applicable for evaluation the effects of all types of solutes. The new temperature-based aquagram calculation method provided further information about the magnitude of the change. In other words, the results were displayed on a degree-Celsius scale that showed how much a given sample would have needed to have been warmed up or cooled down in each of the single coordinates (C01 to C12) to achieve the same results as the actual measurement, while all measurements were performed at precisely the same temperature.

Adding 0.1-M potassium chloride to Milli-Q water resulted in structural changes equivalent to an approximately 0.6 °C temperature increase in the less hydrogen-bonded, and 0.3 °C temperature decrease in the more hydrogen-bonded areas of the OH first overtone spectral region.

The examples presented here confirm the applicability and the additional benefits of the temperature-based aquagram calculation method. They provide a demonstration of the ratio of the different water species existing in different aqueous and biologic systems. Additionally, this new type of aquagram calculation displayed the spectral patterns in a meaningful scale and stable pattern independent of any modification of the evaluated dataset, which gives rise to the opportunity to compare results not only within a single chart, but also across time and different experiments.

This newly developed tool is especially suitable for visualizing water structure evolution and phase transitions, for example, in the food preservation industry, pharmaceutical development, material science and related applications.

The presented new chemometric tool, developed in R-Project, is freely accessible as an R-package from GitHub repository [ 58 ] and can be used in various fields of NIR spectroscopy and water research.

Acknowledgments

Authors are grateful to Professor David Funk for his help to revise the manuscript from language editing and from scientific point of view.

Dataset. Raw spectral data used for the calculations presented in this study. Both data of temperature experiment and potassium chloride experiment is included in this dataset where the column experiment describes which spectra belong to which experiment.

Workflow of the calculation protocol of temperature-based aquagram.

Author Contributions

All authors contributed to this research. The design of the experiments was performed by Z.K., B.P. and R.T., Z.K. and B.P. did the recording and the processing of the NIRS data, as well as the result of the evaluation and implementing the concept in R-project. The manuscript was written by Z.K., B.P., G.B. and J.M., R.T. assisted in writing the study and revised it. Z.K., R.T., G.B. and J.M. contributed to designing the research and revise the manuscript. The work presented in the study was conceived within research projects led by R.T. and Z.K. All authors have read and agreed to the published version of the manuscript.

Authors acknowledge the financial support of the ÚNKP-19–4 New National Excellence Program of the Ministry for Innovation and Technology (Z.K.), János Bolyai Research Scholarship of the Hungarian Academy of Sciences (Z.K.) and by the European Union and co-financed by the European Social Fund through project No. EFOP-3.6.3-VEKOP-16–2017–00005. JM gratefully acknowledges the financial support provided by Japanese Society for Promotion of Science ( {"type":"entrez-protein","attrs":{"text":"P17406","term_id":"114259","term_text":"P17406"}} P17406 ).

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability: Samples of the compounds KCl are available from the authors.

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Article Contents

Water is important, water science on the molecular scale, an emerging frontier, modern water science research.

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Water science on the molecular scale: new insights into the characteristics of water

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Jun Hu , Zexian Cao, Water science on the molecular scale: new insights into the characteristics of water, National Science Review , Volume 1, Issue 2, June 2014, Pages 179–181, https://doi.org/10.1093/nsr/nwt015

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The highest good is like that of water. The goodness of water is that it benefits all the creatures; yet itself does not scramble, but is content with the places that all men disdain. It is this that makes water so near to Tao. —Tao Te Ching

‘Hydrophobic water layer’: water does not wet water. Water shows anomalous behavior on the molecular scale.

Water is a basic component of human existence, and people and ecosystems depend on it. It is one of the most fundamental requirements for the survival of all living things. However, water is a finite resource that has quantitative limitations and qualitative vulnerability. Water shortage is a global concern owing to increasing population, economic growth and climate change. China is facing severe water scarcity. Some experts believe that the water crisis may come before an energy crisis in China. Energy and water are inextricably and reciprocally linked; the production of energy requires large volumes of water (for example, in the USA and China, electricity production requires over 40% of all daily freshwater withdrawals [ 1 ]), and both the treatment and distribution of water depend upon readily available, low-cost energy. More recently, water management for unconventional shale gas extraction has dominated environmental debates surrounding the gas industry [ 2 ].

Water has long been an important topic in science and technology. Water has been intensely studied, and ‘water science’ refers to studies on water and related issues. In the past, water science was concerned with large volumes of water such as the flooding of rivers, atmospheric water evaporation/condensation, freezing/melting of seawater and physical/chemical properties of bulk water in industrial processes. However, modern water science is also concerned with water on the molecular scale. This is because modern science depends on the understanding of matter at the molecular level. Whether it is in the fields of life sciences, materials science or environmental science, scientists realize that answers to their questions are taking place on the molecular scale.

From a molecular point of view, many bulk properties of water are still not well understood. ‘Water is simple but very complex’, Professor Guozhen Yang said in his opening speech at the Water Science Forum 2013, held in Beijing. It is simple because everyone knows that water is H 2 O and many scientists believe that we understand water. However, we know very little about it. Water is not simply a molecule of H 2 O, but a group of H 2 O molecules linked by hydrogen bonds, and this constitutes the most mysterious matter in the world. Water has many anomalous properties. For example, no other liquids are found simultaneously in all three phases: gas, liquid and solid; water undergoes a negative thermal expansion below 4°C; water freezes from the top surface; water O–H stretching vibrations last longer at high temperatures; hot water freezes more rapidly than cold water; high surface tension and small surface potential co-exist in water; and anomalous magnetic and microwave radiation effects occur in water. Recently, it has been revealed that water is more mysterious when confined to an interface, as interfacial water has different properties from the bulk state. Researchers have devoted much effort to understanding interfacial water properties; however, little is known about water interfaces and the structure of water when confined in small spaces.

Recently, there has been a growing interest in water science owing to the great progress in theoretical and experimental tools over the last decade, particularly at the molecular scale. Third generation synchrotron X-ray spectroscopy has revealed the atomic and molecular structures of liquid water and ice. Using scanning tunneling microscopy (STM), one can see single water molecules and water clusters on metal surfaces in a vacuum at low temperature. High-resolution images of the structure of water on electrodes can be obtained routinely by electrochemical STM. Surface force apparatus and atomic force microscopy have been successfully used to measure the subtle interactions between two solid surfaces immersed in water solutions. Sum-frequency generation probes the structures of various water interfaces at the molecular level as well as ultrafast surface dynamics. Infrared, THz and neutron scattering spectroscopy sensitivities have been improved for obtaining a wealth of information. Based on the data obtained by the above advanced techniques and with the help of supercomputers, molecular dynamics and quantum mechanics simulations are able to answer some tough questions about anomalous water behaviors.

Most anomalous water behaviors can be explained by its hydrogen-bonding network. In water, the hydrogen atom attracts a neighboring oxygen atom from another water molecule. This is the hydrogen bond, and its strength is about 23 kJ mol –1 , which is 5 times the average thermal collision fluctuation energy at room temperature, and is far greater than van der Waals interactions. Hydrogen bonds are 90% electrostatic and 10% covalent; they are not too weak and not too strong. Through hydrogen-bonding networks water molecules form water clusters. These water clusters are neither fully ordered nor fully disordered, and fast dynamic changes occur; hence, the structure of liquid water is complicated. Hydrogen-bonding networks are interrupted by surface forces, resulting in changes in the water structure near an interface. That is why interfacial water has different behaviors from its bulk state.

Some properties of water confined in molecular scale dimensions play a crucial role in many important processes and may offer new solutions to water shortages. Water can flow through a hydrophobic nanotube at a fluid rate over 100 times that in bulk channels [ 3 ]. Since the pore sizes in membrane materials used for water treatment can be fabricated on the nanoscale, this finding is important for the future development of membrane materials. Molecularly thin water films exist at air/solid interfaces and ordered structures such as ‘room temperature ice’ form [ 4 , 5 ]. More surprisingly, some researchers reported that water layers on hydrophilic surfaces sometimes behave like a hydrophobic surface that was dubbed ‘hydrophobic water’ [ 6 ]. These unusual properties change the conventional concepts of wetting and friction, inspiring researchers to design and fabricate new materials and devices to manipulate water. These molecular insights into water behavior also influence processes in atmospheric chemistry, soil evolution and rock erosion, and can provide new solutions to many environmental problems and climate changes [ 7 , 8 ]. Traditionally, catalysis has been carried on in a vacuum. In the 21st century, catalysis studies have been started to be performed under atmospheric conditions, and scientists consider that surface and interfacial water play an important role in catalysis [ 9 ]. Water in a cell has different properties from that in a beaker because of crowding and the confined environment. Recent findings about the structures and dynamics of the water hydration layer around proteins and DNA molecules enable a better understanding of protein folding and hence lead to better drug design. Water clusters participate in many biological interaction processes, and not only do they affect biological structures but also provide channels for electron and proton transportation [ 10 ].

Molecular level water science research raises the hope to solve water-shortage problems and provides many challenges for scientists. When we investigate water deeper, we find that water often causes controversies in scientific communities. In history, water's complexity and oddness misled to scientists investigating polywater and developing ‘water memory’ theories. Currently, in water science research almost every important issue is controversial. An important feature of the hydrogen bond is that it is dynamic and possesses direction. What kinds of structures a water molecule constitutes with its neighboring molecules remains a mystery and raised hot debates. It is not clear how hydrogen-bonding networks are disrupted if, for example, water molecules are close to a surface, or if other molecules are dissolved among them. The properties of water/air interfaces are currently debated, and more recently, the observation of interfacial ‘nanobubbles’ with long lifetimes have instilled new enigmas as thermodynamic equilibrium water/gas states are investigated. The mechanism of water splitting on a surface by light, a very promising way to harvest energy from the sun, is still not clear.

‘The importance of water science depends on one's viewpoint,’ Professor Y Ron Shen said in his speech at the Water Science Forum. He indicated that water science on the molecular level is complex, requiring multidisciplinary knowledge. However, difficulties are encountered because of the lack of effective molecular level techniques. Understanding the mysterious properties of water at the molecular level needs theoretical and experimental collaborations, and relating the results to effective ways to benefit society requires multidisciplinary research efforts. Molecular level water science research is an emerging discipline, and Chinese scientists have the opportunity to work in multidisciplinary research projects.

In summary, understanding water and knowing how to manipulate water at the molecular level will offer new solutions to water scarcity problems and save China and the world. Basic and applied water research is important to help build an environmentally sustainable world.

This paper is a collection of views presented at the ‘Water Science Forum, Beijing, June 4–5, 2013’. The authors thank all the attendees, especially Professors Guozhen Yang and Y Ron Shen.

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The shape of water: What water molecules look like on the surface of materials

Trending Research

Impact Science

Understanding the various molecular interactions and structures that arise among surface water molecules would enable scientists and engineers to develop all sorts of novel hydrophobic/hydrophilic materials or improve existing ones. For example, the friction caused by water on ships could be reduced through materials engineering, leading to higher efficiency. Other applications include, but are not limited to, medical implants and anti-icing surfaces for airplanes. However, the phenomena that occur in surface water are so complicated that Tokyo University of Science, Japan, has established a dedicated research center, called “Water Frontier Science and Technology,” where various research groups tackle this problem from different angles (theoretical analysis, experimental studies, material development, and so on). Prof Takahiro Yamamoto leads a group of scientists at this center, and they try to solve this mystery through simulations of the microscopic structures, properties, and functions of water on the surface of materials.

For this study in particular, which was published in the Japanese Journal of Applied Physics , the researchers from Tokyo University of Science, in collaboration with researchers from the Science Solutions Division, Mizuho Information & Research Institute, Inc., focused on the interactions between water molecules and graphene, a charge-neutral carbon-based material that can be made atomically flat. “ Surface water on carbon nanomaterials such as graphene has attracted much attention because the properties of these materials make them ideal for studying the microscopic structure of surface water ,” explains Prof Yamamoto. It had been already pointed out in previous studies that water molecules on graphene tend to form stable polygonal (2D) shapes in both surface water and “free” water (water molecules away from the surface of the material). Moreover, it had been noted that the probability of finding these structures was drastically different in surface water than in free water. However, the differences between surface and free water have to be established, and the transition between the two is difficult to analyze using conventional simulation methods.

Considering this situation, the research team decided to combine a method taken from data science, called persistent homology (PH), with simulations of molecular dynamics. PH allows for the characterization of data structures, including those contained in images/graphics, but it can also be used in materials science to find stable 3D structures between molecules. “ Our study represents the first time PH was used for a structural analysis of water molecules ,” remarks Prof Yamamoto. With this strategy, the researchers were able to obtain a better idea of what happens to surface water molecules as more layers of water are added on top.

When a single layer of water molecules is laid on top of graphene, the water molecules align so that their hydrogen atoms form stable polygonal structures with different numbers of sides through hydrogen bonds. This “fixes” the orientation and relative position of these first-layer water molecules, which are now forming shapes parallel to the graphene layer. If a second layer of water molecules is added, the molecules from the first and second layers form 3D structures called tetrahedrons, which resemble a pyramid but with a triangular base. Curiously, these tetrahedrons are mostly pointing downwards (towards the graphene layer), because this orientation is “energetically favorable.” In other words, the order from the first layer translates to the second one to form these 3D structures with a consistent orientation. However, as a third and more layers are added, the tetrahedrons that form don’t necessarily point downwards and instead appear to be free to point in any direction, swayed by the surrounding forces. “ These results confirm that the crossover between surface and free water occurs within only three layers of water ,” explains Prof Yamamoto.

The researchers have provided a video of one of their simulations where these 2D and 3D structures are highlighted, allowing one to understand the full picture. “ Our study is a good example of the application of modern data analysis techniques to gain new and important insights ,” adds Prof Yamamoto. What’s more, these predictions should not be hard to measure experimentally on graphene through atomic-force microscopy techniques, which would, without a doubt, confirm the existence of these structures and further validate the combination of techniques used. Prof Yamamoto concludes: “ Although graphene is a rather simple surface and we could expect more complicated water structures on other types of materials, our study provides a starting point for discussions of more realistic surface effects, and we expect it will lead to the control of surface properties. ”

Title of original paper: Discovery of new microscopic structures in surface water on graphene using data science

Journal: Japanese Journal of Applied Physics

DOI: 10.7567/1347-4065/ab6564

About The Tokyo University of Science

Tokyo University of Science (TUS) is a well-known and respected university, and the largest science-specialized private research university in Japan, with four campuses in central Tokyo and its suburbs and in Hokkaido. Established in 1881, the university has continually contributed to Japan's development in science through inculcating the love for science in researchers, technicians, and educators.

With a mission of “Creating science and technology for the harmonious development of nature, human beings, and society", TUS has undertaken a wide range of research from basic to applied science. TUS has embraced a multidisciplinary approach to research and undertaken intensive study in some of today's most vital fields. TUS is a meritocracy where the best in science is recognized and nurtured. It is the only private university in Japan that has produced a Nobel Prize winner and the only private university in Asia to produce Nobel Prize winners within the natural sciences field.

About Professor Takahiro Yamamoto from Tokyo University of Science

Takahiro Yamamoto has been with the Tokyo University of Science since 2003, when he joined as a research associate in the Department of Physics. Since then, he progressively climbed until obtaining the title of Professor at the Departments of Liberal arts (Physics) and Electrical Engineering. He now manages his own lab group, who focus on using quantum theoretical simulations to understand the physical properties of materials. In addition, he works at the Water Frontier Science and Technology Research Center, where he is the leader of a research group that aims to understand the properties of surface water through theoretical studies and simulations.

Funding Information

This work was supported in part by Grants-in-Aid for Exploratory Research (Nos. 17H02756 and 16H02079) from the Japan Society for the Promotion of Science (JSPS). This work was also supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) under the Program for the Strategic Research Foundation at Private Universities, 2015–2019.

Media contact

Tsutomu Shimizu

Email: [email protected]

for this article

Published on: Feb 06, 2020

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