Thermal investigation of montmorillonite/BSA by fourier transform infrared spectroscopy measurements¹

1.   Introduction

Many theories have been proposed on the origins of life on earth: in the Russian book of 1924, Oparin hypothesized that simple molecules (CH₄, NH₃) reacted to form small bio-molecules and bio-polymers (nucleotides, peptides,). They, then, evolve in multimolecular systems and, finally, they gave rise to the first forms of life [1]. Later, in a book of 1929, Haldane proposed a similar theory about the origins of life [2]: but, it was Bernal (1951) that suggested that clays had a fundamental role in chemical evolution and in the origins of life due to their ability to absorb, protect from ultraviolet radiation and catalyze the polymerization of organic molecules [3]. Then, Cains-Smith (1982) has proposed that clays can act as genetic candidates [4]. Hence, the inclusion of organic molecules and monomers in the layered structure of clays, such as montmorillonite and kaolinite, would favor the formation and replication of biopolymers such as enzymes and polynucleotides. Several literature works have shown that montmorillonite could be considered the cradle of the evolution of the early life forms on earth [5]–[19], also on the basis of its peculiar interaction with water [20]–[25]. From a general point of view, silicate-based clays [26]–[29] are classified in minerals of type 1: 1, such as kaolinite, and in minerals of type 2:1, such as montmorillonite which is characterized by weak bonds between interlayers [30]–[35] with excess negative charges [36]–[39].

The interlayer space of montmorillonite depends on the degree of hydration of the mineral: by increasing the number of water layers, the crystalline lattice expands (Figure 1); by complete dehydration, however, it loses its ability to expand. In fact, in the presence of water, H₂O molecules dispose themselves within the montmorillonite interlayer space, generating an increased space between the layers.

Figure 1.  Swelling of montmorillonite clay in water.

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Regarding the water absorption capacity, this property is very important for these clays. In fact, clays can absorb or desorb water as a function of changes in the moisture content: as above reported, when H₂O molecules are absorbed, they fill the space between the various layers. Montmorillonite has excellent water absorption properties; however, the interaction between the water molecules and montmorillonite can produce swelling. The absorption of water molecules and the swelling of montmorillonite determine to the formation of hydrated states and can give rose to hysteresis phenomena. The montmorillonite swelling and hydration processes play a fundamental role for a wide variety of engineering applications. Furthermore, the anisotropy of a wide class of clays is reflected in a broad variable range of mechanical properties. The structure of hydrated montmorillonite is shown in Figure 2: this anisotropy of montmorillonite can produce great differences in the values of elastic constants, shear modulus, and Young's modulus. Moreover, these mechanical properties decrease with increasing hydration [40]–[42]. Furthermore, as regards the thermal properties, montmorillonite is a good thermal insulator and, moreover, it allows to increase the thermal stability once added as an additive in many materials. Recent developments in the use of self-assembling supramolecular objects to fabricate innovative well-defined nanomaterials links soft matter chemistry to hard matter sciences [43]–[46] and requires the employment of techniques and approaches that to simultaneously detect the structure re-organization and dynamics at the nanoscale [47]–[49]. This is a significant area of research to produce thermal barrier effects in the structure of the composite material: in fact, the thermal barrier properties of clays are appreciated in many applications involving the use of heat-resistant materials and flame retardants. The nanoclays have been largely studied and used in polymer matrix composites to obtain greater thermal stability and better flame retardancy properties. The variation of thermal expansion, under the effect of heat, for metals, polymers and ceramics was analyzed: generally, the order of magnitude of the thermal expansion in polymers, metals and ceramics can be indicated as follows: polymer > metal > ceramics. This order is based on the values of the linear thermal expansion coefficient in the values range 20–100, 3–20 and 3–5 ppm/°C for polymers, metals and ceramics, respectively [50]. Therefore, a higher thermal stability of montmorillonite allows its use as a filler in polymers to make composite materials with a low coefficient of thermal expansion. However, the improvement of the thermal stability of the polymers requires an increase in the ratio between surface and volume of a material (aspect ratio) and, therefore, can be obtained for values of this aspect ratio higher than 100. Several investigations have been addressed to clarify the role played by ribonucleic acid (RNA) in the early life forms [12],[51]–[64] and on the montmorillonite bioprotective effects on RNA oligomers [12],[65]–[67].

The development of advanced nanomaterials exploit the self-assembly process that involves the combination of non-covalent soft interactions with the multi-functionality of building blocks and provides an excellent strategy for the preparation of novel, advanced nanomaterials with highly controlled properties for biotechnology and material science application [68]–[71]. Previously, it has been highlighted that clays have a high specific surface, cation exchange capacity and absorption capacity and, therefore, they are appreciated for their high absorption capacity of cations including Ag⁺, Zn²⁺ and Cu²⁺. Furthermore, previous research in the literature has shown that clays modified with these cations have antibacterial properties [72]–[77]. As regards, however, the antibacterial properties of montmorillonite modified with Fe³⁺ cations, some studies in the literature have shown effective removal of phenolic organic compounds from wastewater due to the oxidative oligomerization catalyzed on the surface by the same Fe³⁺ saturated montmorillonite [78]–[80]. Moreover, it has been hypothesized that the Fe³⁺ saturated montmorillonite could also be able to eliminate the bacteria present in the wastewater. Although montmorillonites modified with Cu²⁺, Zn²⁺ and Ag⁺ cations show antibacterial activity, the presence of these cations in water could pose a potential risk to public health due to their toxicity at high concentrations. Therefore, it was proposed to use the saturated montmorillonite of Fe³⁺ as a possible alternative for the water disinfection process because iron is an essential element for humans [81]. In summary, the effectiveness of Fe³⁺ saturated montmorillonite for the elimination of bacteria present in secondary wastewater has been demonstrated. So, these experimental results suggest as the montmorillonite modified with Fe³⁺ cations could probably be used as an effective antibacterial material for water disinfection in small plants used for the treatment of drinking water and in large plants used for the treatment of drinking water and wastewater. In this experimental work, samples of Montmorillonite and its mixture with Bovine Serum Albumin (BSA) were investigated as a function of temperature by means of Fourier Transform Infrared (FTIR) spectroscopy technique and Spectral Distance (SD) approach. In order to determine the thermal protective effects of Montmorillonite on a system of biophysical interest, a protein, the Bovine Serum Albumin (BSA) was chosen [82]–[89]. It is a serum albumin protein derived from cows and it has numerous biochemical applications such as ELISAs (Enzyme-Linked Immunosorbent Assay) and immunohistochemistry [90]–[95]. Bovine Serum Albumin (BSA) is a small, soft, stable, non-reactive protein, and it is used as a representative short peptide in the drug delivery system [96]–[101]. It is well known that exist different techniques to investigate the behaviours of proteins, such as X-rays, Nuclear Resonance Magnetic, Neutron Scattering, Dynamic Light Scattering, Acoustic Levitation, Raman spectroscopy and InfraRed absorption [102]–[110].

2.   Materials and method

Pristine montmorillonite powders purchased from Merck (Milano, Italy, surface area 250 m²/g), BSA (purchased from Sigma) and double distilled water were used to prepare the samples. Montmorillonite/water mixtures have been prepared by adding to pure protein double-distilled water (80 wt %Montmorillonite); for Montmorillonite/water/BSA mixtures the concentration was: 80 wt %Montmorillonite/20 wt% (BSA (50 wt%) + H₂O (50 wt%)). Fourier Transform InfraRed (FTIR) spectroscopy allows us to characterize the molecule rotational and vibrational motions. This spectroscopic technique explores 14000–10 cm⁻¹ range of the electromagnetic spectrum, which encloses the Near-IR range (14000 ÷ 4000 cm⁻¹), the Mid-IR range (4000 ÷ 400 cm⁻¹), and the Far-IR (400 ÷ 10 cm⁻¹). FTIR technique is a powerful method to investigate the structural and dynamical properties of materials as well as their dependence on temperature: this technique is based on the analysis of the absorption spectra [111]–[114]. In this experimental work, we collected vibrational spectra by means of the Vertex 70 v spectrometer (Bruker Optics, Ettlingen, Germany) using a Platinum diamond ATR. All spectra were collected by using an average of 96 scans with a resolution of 4 cm⁻¹ in a spectral range of 4000–400 cm⁻¹, from a temperature of 20 °C to 55 °C. The pre-processing data procedure was performed through OPUS software and, then, by means of Matlab environment. Due to the complexity of the investigated systems we prefer to analyze globally the spectral features of the samples by applying an innovative approach consisting in the integrated use of the Spectral Distance and Wavelet Cross Correlation protocols. Such an approach reveals to be very effective since in the present study we focus the attention only on the spectra temperature dependence. On this purpose, to characterize the temperature sensitivity of the analyzed samples, the Spectral Distance approach has been used; this latter is based on the following expression:

where A(ω) represents the absorbance at the frequency ω, T_l denotes the lowest temperature, that is 20 °C and Δω is the frequency resolution of the instrument.

3.   Results and discussion

In Figure 2, the FTIR spectrum of Montmorillonite in the spectral range of 4000 ÷ 400 cm⁻¹ at the temperature of T = 20 °C is reported.

Figure 2.  FTIR spectrum of Montmorillonite in the resolution spectral range of 4000 ÷ 400 cm⁻¹ at the temperature of T = 20 °C.

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Figure 3.  FTIR spectra of the BSA in the resolution spectral range of 4000 ÷ 400 cm⁻¹ at temperature T = 20 °C.

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The most significant peaks are located at ~ 3632 cm⁻¹, i.e. O-H stretching; at ~ 1639 cm⁻¹ that represents the O-H bending (hydration) ; at ~ 1113 cm⁻¹ and 1035 cm⁻¹ make reference to the i-O stretching, out of plane and in plane, respectively. Finally, the peak at ~915 cm⁻¹ denotes the AlAlOH bending, at ~793 cm⁻¹ is situated the tridymite peak and the peak at ~529 cm⁻¹ represents the Si-O bending vibration. Figure 3 shows the FTIR spectrum of BSA in the spectral range of 4000 ÷ 400 cm⁻¹ at at the temperature of T = 20 °C.

For the FTIR BSA spectrum, one of the most important IR spectral feature for the protein are the bands of Amide. In particular, in Figure 3, there are at ~ 3292 cm⁻¹ the Amide A, at ~ 1649 cm⁻¹ Amide I and at ~ 1537cm⁻¹ Amide II. Figure 4 reports the FTIR spectra for Montmorillonite/water mixtures in the spectral range of 4000 ÷ 400 cm⁻¹ and in the temperature range of 20 °C ÷ 55 °C.

Figure 4.  FTIR spectra for Montmorillonite/water mixtures in the spectral range of 4000 ÷ 400 cm⁻¹ and in the temperature range of 20 °C ÷ 55 °C.

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Figure 5.  FTIR spectra for Montmorillonite/water mixtures/BSA in the spectral range of 4000 ÷ 400 cm⁻¹ and in the temperature range of 20 °C ÷ 55 °C.

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Figure 5 shows the FTIR spectra for Montmorillonite/water mixtures/BSA in the spectral range of 4000 ÷ 400 cm⁻¹ and in the temperature range of 20 °C ÷ 55 °C.

As it can be seen, in Figure 5, the typical IR bands of Montmorillonite and the peaks of Bovine Serum Albumin (BSA) are observed. More precisely, by increasing temperature a little decrease in IR band intensity at ~3440 cm⁻¹ of the O-H stretching band is observed; this suggests a dehydration of Montmorillonite. The peaks at ~1649 and at 1537 cm⁻¹ are typical of BSA and can be attributed to the C=O stretching vibration of the peptide linkages; they turn out to be very sensitive to the secondary structural components of the protein. These two peaks decrease with increasing temperature.

Figure 6.  Spectra differences in the temperature range 20 °C ÷ 55 °C.

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Before to proceed with the analysis of data, a pre-processing data procedure was performed by eliminating the background. To better investigate the mechanisms of interactions that occur between Montmorillonite and BSA, the spectrum difference has been taken into account. In particular, from the spectrum of Montmorillonite in the presence of BSA, the spectrum of Montmorillonite has been subtracted the spectrum of Montmorillonite so obtaining the spectrum difference. This procedure was adopted for each spectra of all the investigated temperatures. In Figure 6, the spectra differences, in the temperature range 20 °C ÷ 55 °C, are reported.

The thermal behaviour of the investigated systems was characterized by the evaluation of SD (eq. 1). Figure 7 reports SD as a function of temperature for BSA (green circle) and for the spectrum difference (light blue square) together with their linear fits.

Figure 7.  SD as a function of temperature for BSA (green circle) and for BSA in the presence of Montmorillonite (light blue square) together with their linear fits (continuous lines).

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In order to extract quantitative information, a linear fit has been performed:

By this examination, it results that the slope coefficient value for the spectrum difference, m = 1.82 · 10⁻⁴, is lower than the slope coefficient value for BSA, that is equal to m = 7.08 · 10⁻⁴. This suggests that BSA in presence of Montmorillonite has a higher thermal resistance and for this reason Montmorillonite can be considered as an effective thermal bioprotector.

Another approach to investigate the thermal behavior of Montmorillonite consists in applying the wavelet cross correlation method, that allows to determine, evaluating the wavelet cross-correlation coefficient, r_XWT, the degree of affinity between two signals. Such a method is very innovative and powerful and is employed in several kinds of disciplines such as climate, geoscience, physics, mathematics, finance, engineering science and others [115]–[120]. Let's consider two wavelet transforms, W₁(s, τ) and W₂(s, τ) of the investigated spectra, where s represents the scale parameter (s > 0) and τ denotes the shift parameter, and the two wavelet spectra P₁(s) and P₂(s) [121]–[125]. From a mathematical point of view W(s, τ) is the inner product of the function f(x) and scaled and shifted mother wavelets ψ:

where f(x) denotes the one-dimensional function, * is the complex conjugate, and ψ is the mother wavelet:

then, one defines the wavelet spectrum P(s):

and finally, one determines the wavelet cross-correlation coefficient, r_XWT:

The wavelet cross-correlation coefficient varies in the range −1 ≤ r_XWT ≤ 1; if the value is equal to 1 indicates a positive statistical relationship between the spectra; if the value is equal to 0 no statistical relationship between spectra exists; finally, if the value is equal to −1 a negative correlation between the two spectra is present. In the present study, the spectrum at lowest temperature (T = 20 °C) has been chosen as reference wavelet spectrum both for BSA and for the spectrum difference.

Figure 8.  Wavelet cross-correlation coefficient, r_XWT, versus temperature together with their linear fits.

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Figure 8 shows the evaluated wavelet cross-correlation coefficient, r_XWT, versus temperature for BSA (orange circles) and for the spectrum difference (magenta squares) together with their linear fits.

What it emerges is that r_XWT decreases by increasing temperature following a decreasing linear trend. Also in this case, a linear fit has been performed and the slope coefficient value for the spectrum difference, m = 0.8159 is lower than the slope coefficient value for BSA, that is equal to m = 0.9245. These results confirm that Montmorillonite can be considered as a thermal bioprotector.

4.   Conclusion

The present experimental work has allowed to show the importance of Montmorillonite clay as a thermal bioprotector. FTIR data were collected to study the interaction of Montmorillonite with BSA. SD and wavelet analyses, constitute two effective and innovative approaches for the characterization of the thermal properties of pure BSA and of BSA in the presence of Montmorillonite. Both approaches suggest that BSA in the presence of Montmorillonite has a lower spectral sensitivity when the temperature changes and, hence, the role of Montmorillonite as a thermal bioprotector is thus justified.

Acknowledgment (All sources of funding of the study must be disclosed)

This work is to be framed within the Progetto di Ricerca e Sviluppo ““Materiali di nuova generazione per il restauro dei Beni Culturali: nuovo approccio alla Fruizione (AGM for CuHe: Advanced Green Materials for Cultural Heritage)” Asse II “Sostegno all'innovazione”, Area di Specializzazione “Cultural Heritage” Avviso n. 1735/Ric del 13 luglio 2017-Codice CUP B66G18000500005, codice identificativo Progetto ARS01_00697 nell'ambito del Programma Operativo Nazionale “Ricerca e Innovazione” 2014-2020 (PON R&I 2014-2020).