1. Introduction to Aquaphotomics
Aquaphotomics is a young scientific discipline introduced by Professor Dr Roumiana Tsenkova at Kobe University in Japan in 2005 [1
]. The establishment of a new science came in response to the recognized need in the current state of art for a common platform that can provide integration of knowledge about the water structure and functionality coming from various disciplines and most spectroscopy fields.
Water is the simplest compound and is made of two most common reactive elements. It covers more than 70% of the Earth’s surface, comprises almost 2/3 of human body and is the most abundant molecule of all living cells. From nano to micro, meso, and up to the level of galaxies—water is everywhere. Wherever it is found, there are many phenomena involving it for which the mainstream science still does not have an explanation for. In everyday lives water is the first association to the word “liquid”, and yet liquid water is such an atypical liquid—with behaviors so different from other liquids—that its properties are called “anomalies”. This behavior stems from the capacity of water molecules for hydrogen bonding; if it did not exist, water would be a rather uninteresting material and our world would most likely look profoundly different. The hydrogen bonds connect the water molecules into a dynamic network; a water molecular network, or in other words—into a very complex water molecular system.
The past two decades have seen much progress in water science. Due to the significant role it plays in biological systems, water has received considerable attention. Many interesting phenomena where water is a key player stimulated research across disciplines, revealing the significance of water structure and consequently its functionality in properties of materials or processes such as wettability [6
], biocompatibility [7
], cell communication and carcinogenesis [10
], DNA structure [11
], molecular recognition and communication [12
], protein stability [10
], membrane stability and survival in desiccated state [14
], mechanical properties such as kernel hardness [15
] or mechanical behaviors such as curling of the plant stem [16
]—to list a few. Spectroscopy methods such as X-ray, infrared spectroscopy (IR), THz spectroscopy, near infrared (NIR) spectroscopy and others, using light as a probe, proved to be especially valuable tools for water studies and have contributed immensely to elucidation of various aspects of water systems. In general, water-light interaction over the entire electromagnetic spectrum, significantly contributed to a better understanding of water molecular systems [5
Water molecules absorb radiation over the entire range of the electromagnetic spectrum (Figure 1
). In contrast to mid- and far-infrared, where water strongly absorbs, allowing analysis of only very thin samples, in the NIR part of the spectrum, water absorption is much weaker, therefore offering the possibility of analyzing thicker samples and objects rapidly, in a completely non-destructive and non-invasive manner, and with none or little sample preparation. Using light of the NIR range, it is very easy to acquire spectral data of various aqueous and biological systems in real time without disruption of their state and dynamics. Near infrared spectroscopy thus offers a unique window of opportunities to observe the water molecular network as a scaffold—a matrix of every system of which it is an intrinsic part of—in relation to all other contributing elements and factors shaping the system structure, state and resulting dynamics—without any disruptions.
Aquaphotomics as a science was laid on a foundation provided by near infrared spectroscopy [5
]. The breakthrough knowledge regarding the importance of water stemmed from the observation that NIR spectral data for milk of healthy and dairy animals with mammary gland inflammation (mastitis) differed at water absorbing bands (1440 and 1912 nm) [18
]. The presence of disease in an organism influenced many biomolecules (fat, lactose, proteins, etc.); these changes were subtle and sometimes not even visible in the spectra at absorbance bands related to those compounds. However, all these components exerted an influence on the water structure, and this cumulative effect was observable and measurable at multiple water absorbance bands corresponding to different water molecular species. In other words, the water molecular network changed when the composition of the aqueous system was altered, and this was reflected in water spectral pattern.
This innovative knowledge changed the approach in spectral analysis and paved way for the development of aquaphotomics. Changes in the water spectrum accurately and sensitively reflect the changes of water molecular species, hydrogen bonding and charges of the solvated and solvent molecules. In liquid water, each water molecule forms bonds with neighboring molecules, and can also establish dipoles and induce dipole interactions with other molecules, which gives the water molecular systems a heterogeneous character responsive to physical and environmental conditions [10
]. Specific water species such as free water molecules, dimers, solvation shells and others contribute to the water spectrum in a very distinctive manner [5
]. The water on a molecular level behaves as a collective mirror—its spectrum depicts changes as a response to all internal and environmental perturbations [5
]. Rich experience acquired during many years resulted in a big database of spectra acquired under various perturbations, which revealed information regarding the water molecular system dynamics and the functionality of water in bio-aqueous systems [5
], supporting the recognition of water as an active molecule and a central player in living processes [10
The aim in establishing aquaphotomics as a science on its own, came in response to the recognized need in the current state of art in “omics” disciplines. Despite huge contributions of genomics, proteomics, metabolomics, transcriptomics and etc. to the comprehensive understanding of the principles underlying basic living functions, they all have an approach focused on single molecules and involve extraction procedures with the sample disruption. Biological systems can be studied using a non-destructive and integrative approach based on aquaphotomics, i.e., the interaction between water and biomolecules in which spectroscopic techniques combined with multivariate analysis represent a powerful tool.
Therefore, aquaphotomics aims at integrating and systematizing the knowledge about water-light interaction into a complementary, novel “omics” discipline whose objective is the large-scale, comprehensive study of water, its structure and related functionality. The first step towards this goal is identification of all the absorbance bands corresponding to specific water species. In this way, by knowing what each frequency means in the terms of the water structure, the absorbance bands become like “letters” that could be used to describe the features of aqueous systems. Relating the observed spectral patterns (combinations of water absorbance bands and the intensities of absorbance at these bands) with the observed characteristics or behaviors of aqueous systems, will clarify the functionality of certain water species and allow for future descriptions of the system states and dynamics solely in terms of the water structure. For various systems under various perturbations, aquaphotomics aims to build an aquaphotome, a comprehensive database of water bands and spectral patterns which describe the system and can therefore be used for future evaluations.
Since its establishment in 2005, aquaphotomics has showed steady progress (Figure 2
). From only eight articles published in the first year after it was first introduced, the influence of the general idea and change over the years in the approach of how water is seen and treated in spectroscopy field can be seen. If the current trend continues, the estimate is that in the year 2025, 500 research articles per year can be expected. Through fundamental research, aquaphotomics provided novel insights and a better understanding of the basic phenomena and the role of water. It stimulated research and development of novel signal processing and chemometrics methods for data analysis, and provided a novel common measurement platform for a variety of applications, which led to the development of novel sensing devices and instruments based on water-light interaction.
In the following sections, the key ideas of aquaphotomics, which lead paradigm shift of water seen from passive to active component of bio-aqueous systems and how this affected the basis of novel measurement platform—will be explained. Together with the brief illustrations of major contributions to science so far and through the extensive but not exhaustive list of applications, an overview of the huge technological potential of aquaphotomics will be presented.
3. Aquaphotomics—Innovative Knowledge Leads to Innovative Applications
Being rapid and non-destructive, NIR spectroscopy is a powerful technique whose horizons have been further expanded by aquaphotomics. Since its establishment, aquaphotomics has grown into a multidisciplinary scientific field, encompassing many research areas and providing a common measurement platform for many applications. Using near infrared spectroscopy in aquaphotomics—in comparison to using light of other frequencies—does offer significant advantage of non-destructive evaluation of aqueous systems, which is of special significance for not only exploration of biological systems, but offers immense potential for biodiagnosis and biomonitoring. This region is furthermore an excellent tool for water observation, which provides an enormous amount of information about water molecular structure [5
]. Numerous NIR spectra can be obtained in various conditions and states of the systems (under different perturbations)—all in real time.
The work in such a wide variety of applications, with different systems in different conditions led to two significant breakthroughs in aquaphotomics. The first breakthrough is that water spectral pattern can be used as a collective, integrative biomarker—a descriptor of a system’s state [4
]. The second one is the discovery that the water spectral pattern is related directly to certain functionality of the system. While the first breakthrough is of major significance for applications and provides a novel measurement platform, the second one leads to innovative knowledge of many phenomena. The next sections will illustrate the significance of both.
Contrary to the common understanding of overtone spectroscopy (100 to 1000 times lower absorbance than in the mid-IR range), it has been shown that even very small concentrations of the solutes could be measured with NIR spectroscopy if the aquaphotomics water-mirror approach is applied. Changes in the absorption spectrum of liquid water were used for quantification of the solutes present in water, even when the solutes did not absorb NIR light at all [30
]. For instance, using very robust experimental design, Gowen et al. performed comprehensive aquaphotomics analysis of aqueous salts solutions (NaCl, KCl, MgCl2
) with the aim of establishing limit of detection [35
]. This research demonstrated that the best region for the prediction of salt concentration was the first overtone of water, attaining the prediction error of 500-800 ppm. Similar detection limit (1000 ppm) was reported in a research study that explored quantification of different metals (Cu(II), Mn(II), Zn (II) and Fe(III)) in aqueous HNO3
], while another work reported successful prediction of HIV virus concentrations in plasma with the standard error of 23 pg/ml (ppb level) [60
]. The water-mirror, indirect approach enables measurements of concentrations previously thought impossible to be measured with NIR spectroscopy at ppm and even at ppb levels under certain experimental conditions [23
]. However, if we look beyond the measurements of individual solutes, what these results illustrate is the sensitivity of water molecular network to the changes in its components. The successful applications list measurements of acidity, pH [63
] and effects of mechanical filtration on pure water [64
]. Introducing water spectral pattern as an integrative marker represents one step forward from the detection of individual contaminants in water quality monitoring [65
] or measurements of single, individual biomarkers in disease diagnostics [4
This concept is radically novel, because it shifts the perspective of the definition of water quality by a set of physico-chemical and microbiological parameters to the definition of water quality as a water spectrum within some defined spectral limits. The same is true for disease diagnostics, which for many of diseases, especially in the early stage of development, works with very low concentrations of biomarkers in body fluids or does not even have reliable biomarkers. The spectrum of aqueous system integrates the influence of all single markers into one integrative, holistic marker which is a result of cumulative effect of many components and can easily be monitored in real time. The applicability of the proposed concept was evaluated in water quality monitoring [65
], food quality monitoring [66
] and biodiagnostics [67
]. Using water as a biomarker, the information on the health status of any organism can thus be acquired in real time and non-destructively, allowing the continuous in vivo monitoring of the same sample.
In plant biology studies, aquaphotomics provided a methodology to follow the impact of a virus infection based on tracking changes in water absorbance spectral patterns of leaves in soybean plants during the progression of the disease [69
]. Compared to currently used methods such as enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and Western blotting, aquaphotomics was unsurpassable in terms of cost-effectiveness, speed, and accuracy of detection of a viral infection. The diagnosis of soybean plants infected with soybean mosaic virus was done at the latent, symptomless stage of the disease based on the discovery of changes in the water solvation shell and weakly hydrogen-bonded water which resulted from a cumulative effect of virus-induced changes in leaf tissues. A similar study reported the detection of begomovirus in papaya leaves with an aquaphotomics approach [71
]. Tracking the cumulative effect of various, most likely unknown, biomarkers of viral infection in leaves provided grounds for successful, early diagnosis based on aquaphotomics principles.
Similarly, different water spectral patterns were found in leaves of genetically modified soybean with different cold stress abilities [69
]. This research on the discrimination of soybean cultivars with different cold resistance abilities has proven that resistance to cold stress can be characterized by different water absorbance patterns of the leaves of genetically modified soybean. Different genetic modifications resulted in a multitude of bio-molecular events in response to cold stress, whose cumulative effect was detected as a specific water spectral pattern of leaves; i.e. the higher the cold resistance, the higher was the ability of the cultivar to keep the water structure in less-hydrogen bonded state, providing a supply of “working water” in the conditions of decreased temperature.
In another study, aquaphotomics was applied for exploration of the extreme desiccation tolerance i.e. the ability of some plants—called resurrection plants—to survive extremely long periods in the absence of water and then to quickly and fully recover upon rewatering [72
]. Application of aquaphotomics to study one such plant—Haberlea rhodopensis
—during dehydration and rehydration processes, revealed that in comparison to its biological relative—a non-resurrection plant species, Deinostigma eberhardtii
performs fine restructuring of water in its leaves, preparing itself for the dry period. In the dry state, this plant drastically diminished free water, and accumulated water molecular dimers and water molecules with four bonds (Figure 8
). The decrease of free water and increase of bonded water, together with preservation of constant ratios of water species during rapid loss of water, was found to be the underlying mechanism that allows for the preservation of tissues against the dehydration-induced damages and ultimately the survival in the dry state.
In the medical field, aquaphotomics was proposed for in vivo therapy monitoring of topical cream effects [73
], for monitoring of dialysis efficacy [67
] and diagnosis of several diseases: cancer [67
], diabetes and coronary heart disease [75
]. These applications utilize the concept of a water spectral pattern as an integrative biomarker that offers significant advantage compared to traditional ways of therapy monitoring or diagnostic practices in medicine. For example, monitoring dialysis efficacy is a particularly challenging task that relies on discrete sampling and measurements of only several uremic toxins out of more than 80 currently recognized that contribute to the uremic syndrome (Figure 9
). The NIRS method has already been proposed to measure urea in spent dialysate [76
]. However, urea is only a single marker and its concentration decreases during dialysis, making the detection harder. By using aquaphotomics approach, individual component measurements were replaced by process monitoring [67
]. Instead of measuring waste materials in spent dialysate, their cumulative effect on the water matrix was measured as water spectral pattern changes during the dialysis. In another words, individual component measurement was replaced by monitoring of the process. The water spectral pattern of spent dialysate averaged for all patients after 5, 45, 90 and 135 min of treatment presented as the aquagram in Figure 9
showed, as the therapy progressed there was an increase of free water molecules (1398 and 1410 nm: C5 WAMACS) in the dialysate. In this way, the efficacy of dialysis can be assessed in a simplified way by tracking the changes of the respective dialysate water spectral pattern. The advantage of such an indirect approach of biomonitoring can also be extended to biodiagnostics as the water spectral pattern captures the information regarding all biomolecules that change with the disease—even the biomolecules current science is not aware of.
The works on mastitis [21
] showed that as the various milk components change during the different stages of infection, they influence the water matrix of milk differently. The water spectral patterns of blood, milk, and urine of mastitic cows, revealed that the same water absorbance bands are activated in different body fluids in response to the presence of disease [81
]. Similarly, physiological changes such as ovulation can be detected in various body fluids using the same principles such as in the Giant panda [68
], in the Bornean orangutan [84
], in dairy cows [85
] and in mares [86
Aquaphotomics made a significant contribution to the field of microbiology and food engineering by not only providing a fast and nondestructive analysis, but by contributing to better understanding of the mechanism of action of some microorganisms [87
]. For example, probiotic, non-probiotic and moderate bacteria strains produced a unique water spectral pattern, as shown in aquagrams reported in Figure 10
. Probiotic bacteria strains were characterized by a higher number of small protonated water clusters, and free water molecules and water clusters with weak hydrogen bonds [89
]. The discovery that strong probiotic bacteria produced more free water and less hydrogen-bonded water species, i.e. they break water structures in a way comparable to an increase in temperature, provides novel insight on their mode of action. Moreover, aquaphotomics was able to distinguish a subdivision into two species within one bacteria strain, where conventional PCR analysis was not enough sensitive [90
The aim of studying water interactions on a molecular level was to obtain a better understanding of the relationship between the water structure and a phenomena on a macro scale. For example, one of the novel studies related the sensory texture of apples with particular spectral pattern of fruits: mealy apples had water predominantly in a weakly hydrogen bonded state, while the opposite was true for juicy, firm apples [66
]. Another study related the dehydration band (1398 nm) with physical damage in mushrooms [45
]. Similarly, wheat kernel hardness was related to specific water absorbance bands (1366 and 1436 nm) [15
]. Usually, food texture is not considered a property that stems from certain water structure; however, the above-mentioned works revealed that water structure change with texture. Further studies are needed to better understand the relationships between the water spectral pattern and pectin metabolism in horticultural products.
Interesting findings were obtained in applications of aquaphotomics for basic studies of interaction of biomolecules and water. For example, although many spectroscopic studies have been conducted on glucose, few studies have been carried out on the anomers of glucose despite the fact that spectra—as well as chemical and enzymatic reactions—depend on the specific molecular structure. What aquaphotomics study of glucose isomerism [91
] found is that the absorbance band at 1742 nm possess the potential to distinguish glucose anomers qualitatively and quantitatively. What is conventionally regarded as the first overtone of the C-H stretching mode, was confirmed to not be related to glucose—but to water [92
]. Through work in the field of protein-water interactions, aquaphotomics provided insight into their dynamics and the significant role water plays in their functionality. In a study of prion protein isoforms [47
], aquaphotomics analysis of Mn and Cu prion isoforms in water solutions revealed that while binding of copper results in increased protein stability in water, the binding of manganese resulted in less stability—which led to fibril formation, responsible of neurodegenerative disease. The fact that the entire process of protein structural changes in aqueous systems can be monitored indirectly through the water absorbance pattern of the protein solution, was demonstrated in a study of amyloid protein—another protein involved in pathogenesis of neurodegenerative diseases [71
]—as well as ovalbumin [51
Aquaphotomics studies on water-material interaction hold great promise in understanding some of the very complex properties that are of interest for many applications, such as wettability or biocompatibility. A study concerned with investigation of an excellent wettability of titanium dioxide reveled the importance of water species ratios [6
]. More recent studies exploring the state of water in hydrogel materials of soft contact lenses [94
] revealed that the water spectral pattern holds information even about the state of polymer network and protein deposits on the surfaces of worn contact lenses. Other aquaphotomics studies showed how nanomaterials shape the water matrix, as in the case of fullerene-based nanomaterials that act as water structuring elements when present in very low concentrations [73
]. In nanotechnology and nanomedicine, aquaphotomics could lead to novel findings due to the fact that with decreasing size, the available active surface interacting with water playing a significant role increases.
Since its establishment, aquaphotomics has grown into a large, multidisciplinary scientific field, encompassing many research areas and providing common measurement platform for many applications. Table 2
provides an idea of possible fields of applications of aquaphotomics coupled to NIR spectroscopy. These works illustrate the great versatility of this technique and can hopefully inspire novel research and application ideas.