Description of New Bioelectromechanical Properties in Alginate : An Insight with a Computer Simulation

Alginate biopolymer from Tropicalgin C302245 was studied by means of piezoresponse force microscopy imaging, scanning electron microscopy, powder X-rays, infrared spectroscopy and computer simulations. Local piezoresponse force microscopy images show possible ferroelectric domains detected in the out of plane mode and these results are confirmed by the second harmonic generation analysis. Alginate powder is composed by diatom frustules containing a cristobalite-like compound, amorphous silica and chitin. The experimental results are explained by MM+ and PM3 computer simulations that establish that the self-assembly of the alginate molecules enhance the polarization increasing the molecular collective dipole moment. Alginate molecular properties might open interesting possibilities for organic technological applications.

Advances in Materials Physics and Chemistry, 7, 334-346.https://doi.org/10.4236/ampc.2017.78026Advances in Materials Physics and Chemistry related functions as stated by Galvani and Volta [1].Voltage-gated signaling [2] is found in a wide distribution of taxa, from bacterial biofilms [3], plant tonoplasts [4] up to mammal neurons [5], thus the relevance of the energy from potentials and moving electrons must be a prerequisite for cellular response.At the molecular scale, electric potentials might stimulate changes in the conformational features of peptides [6] and in the classic experiments of the synthesis of amino acids [7] electric potential was of main relevance.The Mexican scientist Alfonso Luis Herrera noting the importance of the electricity in molecules, stated "(...) Nevertheless, the modern and fertile ideas about the ionization of molecules tend to give prominence to the production of physico-chemical phenomena by direct or indirect, mediate or intermediate electric forces and actions.The true philosopher is always ready to believe these transformations of energy and the true naturalist expects and provides the constant transformation of species" [8].Other works analyze the main role of bioferroelectricity in molecular order and functionality of biological membrane channels [9] [10].Many biologically-originated materials possess piezoelectric properties at the nanoscale [11] such aspolymers, biomolecules and bio-structures already published [12] [13] [14] [15] [16].It seems that molecular ferroelectricity is relevant in biology [9] [14] [17] [18] [19] thus, rising an enquiry: whether or not the biomolecular electrical properties are relevant in biological activity, coordinating an additional molecular selection and self-assembly?If so, that assumption might contribute to the understanding of the biological functionality [20].Piezoelectricity was discovered on wood by Shubnikov in 1946, and afterwards in other organisms tissues (teeth, horns, and cartilages) [21] and soft tissues (human pineal gland [22]), as well as on the micro and nanoscopic scales by using the piezoresponse force microscopy (PFM) [23].The piezoresponse force microscopy is the most descriptive method to get insight into the piezoelectric and ferroelectric phenomena at the nanoscale [23] in different materials.Nevertheless, the comprehension of these phenomena at the molecule scale has not been explained accurately, more specifically, why polymers present this effect [24].
Alginatesas biopolymers in bacteria seem to play a structural function, affording mechanical strength and flexibility, meanwhile the molecule grasps water [25].Alginates are polymers of two alternating acidic monomers, α-L-guluronic (G) and β-D-mannuronic (M) organized in different oligomers rich in G units and blocks rich in M units.The mechanical properties of alginates depend on the G: M ratio and has a relatively easy industrial production [26].For this reason, and its biocompatibility, alginate has emerged as biologically relevant hydrogel material for drug delivery [27].In other studies, alginate is used in inkjettechnology to produce three dimensional assemblies mediated by different voltages [28] [29] what makes us to consider the feasibility to possess some electromechanical features.Hence, it is a necessity to get an insight into the activity of the alginate under the electric field at the molecular-scale.Here, we report, the potential nanoscale bioferroelectricity in the biopolymer alginate (Figure 1).In biological self-assembled macromolecules, it is possible that different molecule-Advances in Materials Physics and Chemistry scale mechanisms activate ferroelectric memory effects [30] [31] [32].These properties will be useful to design green computer memories, biosensors and harvest energy devices [30].The observed promising ferroelectric behavior adds a new important functionality to this well-known biomaterial.

Materials and Methods
The samples were the Alginate single dose Tropicalgin C302245, (lot 150884, Zhermack Clinical, Rome, Italy) commercially obtained.Directly from the package, the samples were used with no further treatment.

Scanning Electron Microscopy (SEM)
All the samples were fixed in a sample holder using only carbon ribbon, without any additional preparation.Scanning electron microscopy (ultra high resolution JSM-7800 FS chottky Field Emission Scanning Electron Microscope) instrument was used.We performed the measurements in a vacuum of 0.55 MPa, with an acceleration voltage between 2 -5 kV.

X-Ray Diffraction (XRD)
For the XRD study, powder samples were put in a Phillips X'pert instrument with a Cu-Kα1 radiation at room temperature.We performed the data collection using a step size of 0.5˚ over 10˚ -80˚ 2θ range (Figure 2).

Piezoresponse Force Microscopy (PFM)
Samples were cast and fixed with no further treatment to be visualized by the     GAMESS.The molecules were edited from HyperChem8.0.1 and afterwards in Avogadro 1.2.0 [36] and exported for GAMESS software [37].We used the PM3 semi-empirical calculations and a run type of Optimization with Restricted Hartree-Fock (RHF) molecule charge 0 and spin multiplicity 1.

Results and Discussion
Alginate (Tropicalgin C302245) powder is composed by diatom frustules (Figure 1) seeming to have a highly crystalline material (Figure 2) resembling   other studies containing opal-cristobalite of fossil diatoms [38] [39].X-ray structural parameters from alginate powder shows a very crystalline material corresponding tocristobalite crystalline solid (Figure 2).In the case the IR spectra, they show the characteristics set of SiO 2 bands from biosilica [33], alginate [40] [41] and chitin [42] [43].Concretely, the band at 3437 cm −1 is due to the stretching vibration of -OH group and correspondingly, the IR band at ca.1617 cm −1 is due to the adsorbed water molecules.A strong and broad FTIR band at 1170 cm −1 with a shoulder close to 1200 cm −1 is usually assigned to the Si-O-Si asymmetric stretching vibrations, whereas the IR band at 793 cm −1 can be assigned to Si-O-Si symmetric stretching vibrations [44].Alginate spectrum demonstrates the characteristic absorption bands of its polysaccharide structure (inset in Fig- ure 3), symmetric 1414 cm −1 and asymmetric 1600 cm −1 (C-O stretching) from the carboxylate salt groups.Conversely, the bands at 1126 cm −1 (C-C stretching), 1021 cm −1 (C-O-C stretching) and 947 cm −1 (C-O stretching) are screened by the SiO 2 bands.The partially masked chitin bands at ca. 3250 cm −1 , (amide I) ca.1650 cm −1 and amide II ca.1550 cm −1 (in Figure 3, vertical gray lines in inset) confirms the presence of this molecule in diatom biosilica as described already [45].
The AFM allows for a non-destructive inspection of the surface of the sample and the direct monitoring of possible domain structures by means of PFM (Figure 4(a) and Figure 4(b)).This procedure allows studying the local properties such as polarization reversal.Piezoresponse force microscopy in the resonance mode was used to obtain evidence of the ferroelectric behavior of alginate (4B).To determine the amplitude of the AC voltage used in the PFM measurements, the first and second harmonics of the alginate sample were measured by the application of Vac = 1 Vpp over the sample.Clearly, the first harmonic is greater than the second one (Figure 4(a)).These results confirm that alginate might present ferroelectricity [46].The piezoresponse images (Figure 4(c) and Figure 4(d)), were obtained by applying 1 Vpp at 71.5 kHz on a 500 nm by 500 nm square area.The PFM phase (Figure 4(d)) zones present similar topographical heights (Figure 4(b)) although exposing opposite polarization, defining that the observed contrast is due to the changing orientation of the dipole moments.These results agree with theoretical calculations shown later.However, by using Vac = 2 Vpp, displaying a clear topography (Figure 4(F)), the second harmonic is higher than the first one (see Figure 4(e)) thus, presenting no domain contrast in amplitude [47] [48] (Figure 4(g) and Figure 4(h)).The alginate dipolar direction might correlate to the internal rotation of the dipole moment-related chemical groups in asymmetric carbon atoms in the polysaccharide [48].Figure 4, shows the poled sample with areas with different polar phases that are distributed randomly along the alginate sample.The switching contrast (Figure 4(c) and Figure 4(g)) does not follow directly the topographic aspect, indicating that the rearrange effect might be due to the intrinsic ferroelectric properties of the alginate and not induced from the surface electrostatic effects.Our result agrees with other biopolymers [49].The computational molecular modeling of the different alginate units was performed to understand, at the molecular scale, the effects of the self-assembly on the dipole moments.An isolated monomer was optimized, and subsequently covalently linked to other monomers (up to 5 in Figure 5(a) and Figure 5(b)).
The next procedure was to put to interact the 4 up to 10 molecules (Figure 5(c) and Figure 5(d)).Here, the models were studied by the geometry optimization of the total energy, and molecular dynamics cycles run using molecular mechanics (MM+) method in combination with a semi-empirical method PM3 (in RHF).
This precedent allows us to further simulate in the future, the self-assembly of alginate chains, considering those stabilizing ions and molecules.

Conclusion
The alginate powder presents a clear content with different diatom species, together with the molecular assembly (Figures 1-3).The molecular structure resembles the SiO 2 biosilica with features of a crystalline cristobalite-like solid (Figure 2 and Figure 3).Regarding the PFM imaging and switching behavior, our findings suggest that alginate presents a ferroelectric effect and an efficient optical second harmonic generation.The biophysical purpose or aim of this organic-inorganic ferroelectric component is far away from our study, although brings a possible molecular transducer and biocontrol for detection of local electrostatic charges.This kind of polymer can be easily applied in different bionanotechnological instruments developing electromechanical biocompatible systems.The direct measurements of the spontaneous polarization with the PFM in

Figure 1 .
Figure 1.(a) SEM microphotographs showing the morphology of the diatom from alginate powder Tropicalgin C302245 and (b) the molecular structure used for the computer simulations.

Figure 2 .
Figure 2. X-ray diffractogram of the alginate powder sample where the crystalline material and amorphous organic biopolymeric (background) phase are identified.The peak height reflects aqualitative increase in size of the biopolymer-associated crystallites.

Figure 5 .
Figure 5. MM+ force field results show a loss in general stability values in the alginate molecules.A saturation region in the stability (in (a) at four to five units) through the augmentation of units is seen.In (b) an increase in the dipole moment is seen at the value of 4 units.The simulation of the assembly of different chains ((c), (d)) decreases the stability of the system such as in the increase single chain molecular mass ((a), (b)).

Figure 6 .
Figure 6.PM3 semi-empirical results show an increasing stability (decreasing values) of energy in (a) in the growth of an alginate chain and an increase in the dipole moment (b) and a reduction in the heat formation.

Figure 7 .
Figure 7. PM3 semi-empirical resultsin GAMESS (dotted lines) show decreasing energy values (a) when increasing the monomer residues and an increase in the dipole moment (b) and a reduction in the heat formation values (c).These results contrast with the experimental values (lines).

A.
Heredia et al.DOI: 10.4236/ampc.2017.78026341 Advances in Materials Physics and Chemistry Figure5(d)).In contrast, PM3 semi-empirical method in HyperChem, partially agrees with the MM+ in the enhancement of the dipole moment values when increasing the molecular size of the chain (Figure5(b)) thus, making suitable that the increase in the alginate single chains molecular size, rises the flexibility of the molecules, yielding higher dipole moment values (Figure6).PM3 semiempirical simulation of alginate chain growth in GAMESS software, results in similar behavior to those obtained by HyperChem software although with different values.The most similar ones are the dipole moments in both software (Figure6and Figure7).
PM3 semi-empirical values of the assemblies of different number of alginate chains (not shown) show increasing positive energy (loss in general stability and in the ΔH) what agrees with the fact that alginate molecules stabilize through the