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# Biomimetic mineralization of diatom exoskeleton for sustainable pH-sensitive colored chitosan/silica hybrid self-supported films

Sandra Castanié, Laurent Billon

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Sandra Castanié, Laurent Billon. Biomimetic mineralization of diatom exoskeleton for sustainable pH-sensitive colored chitosan/silica hybrid self-supported films. Materials & Design, 2025, 250, pp.113571. 10.1016/j.matdes.2024.113571. hal-04883663

HAL Id: hal-04883663

https://univ-pau.hal.science/hal-04883663v1

Submitted on 13 Jan 2025

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# Biomimetic mineralization of diatom exoskeleton for sustainable pH-sensitive colored chitosan/silica hybrid self-supported films

Sandra Castanié<sup>a,b</sup> <sup>Δ</sup>, Laurent Billon<sup>a,b,*</sup>

## ARTICLE INFO

**Keywords:**

Hybrid films

Chitosan/silica

Bio-inspired material

Diatoms

pH sensor

Alizarin organic dye

## ABSTRACT

A gentle bio-inspired approach to the biosilicification occurring in diatom frustule is proposed to develop, at room temperature and without addition of a co-solvent, self-supported hybrid organic/inorganic films with pH-sensitive properties. It was found that chitosan (CS) biopolymer, a polysaccharide possessing numerous amine groups (–NH₂) is capable of mimicking the action of the silaffins proteins of diatom frustule in the polycondensation process of the TetraEthoxyOrthoSilicate (TEOS) silica precursor to form a cohesive self-supported hybrid film under soft conditions in aqueous media. Thanks to its good film-forming property, hybrid chitosan/silica (CS/SiO₂) films have been elaborated varying the amount of TEOS to study their optical and mechanical properties. It is shown that increasing the amount of TEOS up to 90 wt% improves the transparency, strength and stability of the films due to the interpenetration of the chitosan and silicaq networks. The incorporation of sustainable pH-sensitive alizarin dye during the elaboration of the films does not affect their mechanical properties whatever the composition. Moreover, these colored hybrid materials show good pH-sensitiveness under basic environment. Therefore, thanks to their antimicrobial properties and their pH-sensitivity, such self-supported films could be designed for applications such as smart sustainable food packaging and food degradation indicator.

## 1. Introduction

Biomimicry, or bio-inspiration, which involves observing Nature and copying it or part of it to develop human concepts, technologies and designs, is an approach that has already led to the development of high-performance technologies, and that finds numerous applications in fields such as physics and chemistry [1–6], energy [7,8], medicine [9–12], architecture and design [13], and robotics [14,15].

Taking inspiration from Nature to adopt a biomimicry approach is also an exciting challenge to aim to a more sustainable world. Natural materials can exhibit remarkable properties made of relatively weak constituents due to performant hierarchical structures and hybridation between inorganic and organic constituents. In marine organisms, wonderful performing materials are obtained through very soft conditions of temperature, concentration, or pressure. For instance, nacre from mollusks shells exhibits a remarkable strength and toughness while composed of greater than 95 % aragonite, a brittle ceramic, but

incorporating 5 % of soft biopolymer into a hierarchical structure. Nacre from Abalone shells is ~1000 times tougher than pure aragonite [16,17]. Diatoms are also great examples of inspiring materials due to their original shell structure. Made of transparent amorphous silica, the frustule, i.e. the shell, presents a hierarchized double porosity, mainly micrometric but with nanometric pores at the bottom of the micropores, which insures protection to living cell while allowing nutrients and UV to enter for feeding and photosynthesizing [18]. The process of biosilicification leading to the specific frustule structure involves organic parts for the polycondensation at room temperature of silica dissolved in water. This polycondensation is driven by the complexation of silicic acid Si(OH)₄ by amine groups –NH₂ present in great number in proteins called silaffins, to form the diatom cell based on an organic/inorganic hybrid material [19–21].

With the view to get inspiration from these natural and soft processes to develop performing materials, to resort to biopolymers and organic/inorganic hybridation appears to be a good and sustainable strategy

<footnote><sup>a</sup> Bio-Inspired Materials Group: Functionalities & Self-Assembly, E2S UPPA, 64000 Pau, France</footnote>

<footnote><sup>b</sup> Université de Pau et Pays de l'Adour, E2S UPPA, CNRS, IPREM UMR 5254, Institut des sciences analytiques et de physico-chimie pour l'environnement et les matériaux, 64000 Pau, France</footnote>

<footnote>* Corresponding author at: At Bio-Inspired Materials Group: Functionalities & Self-Assembly, Université de Pau et Pays de l'Adour, E2S UPPA, CNRS, IPREM UMR 5254, Institut des sciences analytiques et de physico-chimie pour l'environnement et les matériaux, 64000 Pau, France.

E-mail address: laurent.billon@univ-pau.fr (L. Billon).</footnote>
followed by an important scientific community [22–25]. In the partic-
ular case of the biosilicification process occurring in diatom frustules,
the biopolymer candidate must have numerous amine groups –NH₂ able
to mimic the action of silaffins in the polycondensation of silica pre-
cursor into a silica film [21]. Among biodegradable polymers, chitosan
(CS) is a linear polysaccharide composed of randomly distributed β-(1
→ 4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucos-
amine (acetylated unit). With its protonated amine functions at slightly
acidic pH, it appears to be of interest to mimic the silica complexation
process of diatom. Moreover, chitosan presents the advantages to be one
of the most abundant natural food sources, to be biocompatible and
nontoxic, to be a good film-forming, to resist to the soft heat and to have
a good adsorption capacity. These properties permit to find a lot of
applications in various fields such as medicine, cosmetics, photography,
depollution, or food packaging [26]. However, the poor solubility of
chitosan limits its application and processing convenience.

Chitosan/silica (CS/SiO₂) hybrids have recently received much attention due to improvement in chemical and mechanical properties of chitosan thanks to the addition of silica nano-particles [27–30]. Moreover, the introduction of silica into biomaterials can increase its oxygen permeability, biocompatibility and biodegradability [31].

The sol–gel process is a well-known technique in soft chemistry permitting to combine organic and inorganic parts to develop materials with attractive properties. Here, considering the good film-forming property of chitosan, the sol–gel route can be an effective way to develop interpenetrated chitosan/silica hybrid films with enhanced chemical and physical properties.

If some papers describe the formation of CS/SiO₂ hybrid films, these last ones are elaborated by simple mixing of silica nano-particles with chitosan, or requiring temperature or co-solvent [32–34]. Nevertheless, to mimic the soft conditions and the chemical reaction which takes place during the process of biosilicification of diatoms, the process has to occur in aqueous media under soft conditions. A such bio-inspired approach was recently described by Larrieu et al. [35] to form CS/ SiO₂ films that mimic the composition of the diatom frustule.

Moreover, due to the soft conditions, the sol–gel process was also described to permit the incorporation of additives such as pigments and dyes. Colorimetric pH indicators are of great interest in many fields of science due to their easy production in a wide range of colors and a broad range of applications [36,37]. Synthetic dyes such as bromomethyl blue, bromocresol green, cresol red or methyl red are commonly used in the industry of pH-sensitive indicators [37–40]. However, they present an important risk for health and environment due to their toxicity, carcinogenicity and poor degradability [41]. In the last decade, renewable materials have attracted more and more attention and efforts are underway to replace toxic synthetic chemicals with natural or sustainable compounds. As coloring agents, natural dyes have been used for thousands of years but they present the disadvantages to be instable and to rapidly degrade and fade under temperature and light [42]. However, the last years have seen an important investment of scientists in the development of stabilized organic dyes within inorganic supports. The inorganic matrix offers high thermal stability, mechanical strength and solvent resistance, while the organic component provides coloring and functionality [42,43]. Among the natural organic dyes, anthocyanins and anthraquinone pigments are of interest for their pH-sensitiveness. These class of molecules are commonly obtained from fruits and plants such as blueberries, sweet potatoes, red cabbage, madder [44]. Alizarin (1,2-dihydroxy-9,10-anthracenedione, AZ) is considered as one of the most popular and stable dyes due to its high resistance to light degradation. It has been widely used in the painting and fabric industry [45], in dye-sensitized solar cells [46], as marker of calcium biochemical assays [47], or as an indicator to monitor food quality [48,49].

Herein, we propose to highlight the bio-inspired and sustainable approach mimicking the biosilicification occurring in the diatom frustule to design pH-sensitive self-supported and flexible chitosan/silica/ dye hybrid films via a simple sol–gel aqueous route in soft conditions.

The incorporation of bio-sourced alizarin and of various silica contents
in their composition was considered. Chemical/structural character-
ization, optical/film-forming properties and pH-sensitiveness of the
films were investigated.

This approach in aqueous media under soft conditions represents a
sustainable route to develop cohesive self-supported hybrid silica films
with designed properties based on chitosan and in-situ generation of
silica.

## 2. Materials and methods

2.1. Raw materials

Chitosan powder (DDA ≈ 82 %, Mw = 50–150 kDa) was purchased from GTC Bio. Tetraethyl orthosilicate (TEOS, 99 %) and anhydrous magnesium sulfate (MgSO₄) powder were purchased from ABCR. Acetic acid (99 %), hydrochloric acid (37 %), dehydrated calcium chloride (CaCl₂), sodium chloride (NaCl), potassium chloride (KCl) and alizarin dye (AZ) were purchased from Sigma-Aldrich. Magnesium chloride hexahydrated and potassium hydroxide (KOH) were purchased from Merck. Buffers were purchased from Carl Roth. Scheme 1 presents the chemical formula of chitosan, TEOS and alizarin.

## 2.2. Hybrid films preparation

2.2.1. Chitosan/silica hybrid films

Chitosan/silica hybrid films were prepared in soft conditions using
sol–gel route with TEOS as source of silica. As chitosan is soluble in acid
water, CS powder was dissolved in a 2 wt% acetic acid solution (2 g of
acetic acid for 98 g of MilliQ water), to obtain a 4 wt% solution, and was
stirred at room temperature for 48 h until complete dissolution. The pH
of the CS precursor solution was measured by pH-meter equal to 4.3 at
21 °C. TEOS was diluted in MilliQ water to obtain a 4 wt% solution. The
pH of the TEOS precursor solution was measured by pH-paper equal to
about 5.

The two precursor solutions were then mixed together varying the
amount of TEOS from 0 wt% to 90 wt%. The different mixtures were
stirred for 30 min at room temperature and the pH of the solutions was
evaluated by pH-paper. Then the solutions were casted onto poly-
carbonate plates, or glass or quartz microscope slides and placed under
fume hood to dry out. After 24 h, dried cohesive films of 10–150 µm
were obtained.

2.2.2. Chitosan/silica/alizarin hybrid films

pH-sensitive CS/SiO₂ hybrid films containing alizarin dye were
synthesized following the same procedure. Films were obtained by
adding AZ powder at a concentration of 4x10⁻⁴ M with the different CS/
TEOS mixtures. AZ concentration was chosen in accordance with the
poor solubility of the dye in acid media. Before stirring for 30 min, the
mixtures were placed 5 min into ultrasounds to avoid alizarin aggre-
gates. The solutions were then deposited onto polycarbonate plates, or
glass or quartz microscope slides and placed under fume hood to dry out.

2.3. Characterization methods

2.3.1. Fourier-transform infrared (FTIR) spectroscopy

The structure of the hybrid films was examined by Fourier-transform infrared (FTIR) spectroscopy (Model Thermo Fisher Nicolet IS50). The spectra were recorded in attenuated total reflectance (ATR) mode and the wavelength range was between 4000 and 500 cm⁻¹. For the analysis self-supported films were prepared.

2.3.2. Solid-state nuclear magnetic resonance spectroscopy

¹³C and ²⁹Si solid state nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AVANCE III 500WB spectrometer. Prior to the measurements, the films were cryo-grinded to obtain flakes of
<figure><img src="image_4.png" /><figcaption>Scheme 1. Chemical formula of a) partially deacetylated chitosan, b) TEOS and c) alizarin.</figcaption></figure>

maximum 0.5 mm in width. ¹³C spectra were recorded with a 4 mm triple X/Y/Z probe using the CP MAS technique and the adamantane reference (28 ppm). The number of scans performed was 2200, with a recycle delay of 7 s and a contact time of 2 ms. ²⁹Si spectra were recorded with a 4 mm double H/X probe using the HPDEC technique and the octakis(trimethylsiloxy)silsesquioxane (Q8M8) reference at 12.4 ppm. The number of scans performed was 500, with a recycle delay of 150 s.

### 2.3.3. Scanning electron microscopy + Energy dispersive spectroscopy (SEM-EDS)

SEM images of the films' surfaces were recorded on a MEB-FEG Apreo 2 @TFS using an accelerated voltage of 2 kV. All samples were sputtered with 4 nm of platinum before analyses using a high-resolution sputter coater Cressington 208HR. Chemical analysis was recorded using a Bruker EDS XFlash 61100 at an accelerated voltage of 5 kV.

### 2.3.4. Atomic force microscopy (AFM)

AFM adhesion images were recorded using a Bruker AFM Multi-Mode8® on PeakForce QNM mode (ScanAsyst-Air probe), at 2 kHz. For these analyses the films were deposited on a silicon wafer.

### 2.3.5. Mechanical properties of films

Mechanical properties of hybrid CS/SiO₂ films with and without AZ were determined at room temperature using a universal materials tester Alliance RT/50 (Material Testing Systems) equipped with pneumatic jaw. Measurements were performed with a 50 Hz acquisition speed and a 20 mm/min speed test. Previous to the tests, all samples were dried under extractor hood at room temperature during 2 days and stored in petri boxes. The sample thickness was measured at least 3 times randomly using an electronic digital micrometer with a resolution of 0.0001 mm. For the thickest sample (100 % chitosan) the variation of thickness did not exceed 0.036 mm. For the thinnest (90 wt% TEOS), the variation did not exceed 0.001 mm. Between the different compositions, the thicknesses varied from 0.01 for the samples with 90 wt% TEOS to 0.121 mm for pure chitosan films. All the films were cut into rectangular shapes (80 x 10 mm) to test the tensile strength (TS; MPa) and the elongation at break (EAB; %). TS and EAB were calculated using Eq. (1) and Eq. (2), respectively. A minimum of five specimens were tested for each composition. The results given are the average and deviation of the different tests made for a same composition.

$$TS(MPa) = F_{max}/S \qquad (1)$$

where $F_{max}$ is the maximum load, and S is the cross-sectional area of the film.

$$EAB(\%) = (L - L_0)/L_0 \times 100 \qquad (2)$$

where L is the length at which the film breaks, and $L_0$ is the initial length of the sample.

### 2.3.6. Stability into water solutions and water uptake

The stability of the CS/SiO₂ and CS/SiO₂/AZ hybrid films was studied into two different solvents. MilliQ water (pH = 6.4) was used in comparison with an artificial sea water prepared according to the Roscoff station protocol. Composition is given in the Supporting Information Table S1. The samples were immersed into aqueous media for

48 h to appreciate their stability. Water uptake tests were performed on films oven dried at 40 °C for a night and weighed (W1). The samples were then immersed in MilliQ water (test 1) and in artificial sea water (test 2) at room temperature for 1 h to reach the equilibrium. Excess water was removed with filter paper from the swollen saturated films, which were subsequently weighed for the determination of wet weight (W2). The samples were then put back into water for 1 h to repeat the test. 7 measurements were performed. Water uptake (W) of the hybrids was obtained according to the following equation:

$$W(\%) = \frac{w_2 - w_1}{w_1} \times 100\% \qquad (3)$$

### 2.3.7. Contact angle measurements

The apparent contact angle measurement was realized with an optical device (GBX contact angle meter, DGD Fast/60) which allow to determine the contact angle of a droplet with the surface. A drop of MilliQ water or artificial sea water was deposited on a sol-gel coated glass microscope slide. Final values are the average of 4 replicates obtained at different spots.

### 2.3.8. Optical properties of alizarin and hybrid films

The optical properties of the materials were studied by UV-visible spectroscopy on a Shimadzu UV-21021PC spectrometer. Transmittance spectra (300–800 nm) were recorded on CS/SiO₂ hybrid films to evaluate the effect of SiO₂ ratio on the transparency of the films. Absorbance spectra (300–800 nm) of AZ solutions and AZ-containing hybrid films were performed to characterize their pH-sensitivity.

### 2.3.9. Az-containing hybrid films pH-sensing experiment

The pH-sensitivity of the hybrid films containing AZ was evaluated by UV-visible spectroscopy either after exposure to acid and basic atmospheres, or by deposition of acid and basic drops to mimic liquid projections. The films were elaborated onto quartz microscopy slides to avoid deformation in contact with the liquid.

For the tests under atmosphere, the films were either first exposed to an acid atmosphere created by a solution of HCl at pH 5 for 24 h. Then, the same films were exposed to a basic atmosphere for another 24 h (KOH pH 12). Or, they were first exposed to a basic atmosphere (buffer pH 10) for 6 h, then to an acid atmosphere (buffer pH 3) for 24 h. To create the desired atmosphere, the solution was place in small caps and put together with the films in a close petri box. Previously to the experiment, the UV-vis spectrum of each films was registered as reference. Then, after each step, the corresponding UV-vis spectrum was recorded.

For the tests by deposition of acid and basic drops, we proceeded first by the exposure to the acid solution (HCl pH 5). After 1 min, the excess of liquid was removed with a filter paper and the UV-vis spectrum was recorded. Then, multiple drops of a basic solution (KOH pH 10) were deposited on the surface of the film to “wash” the traces of acid before recording the next spectrum. The procedure was repeated a second time and finished with the deposition of acid drops (Table S2).
# 3. Results and discussion

## 3.1. Nature of interaction between silica and chitosan networks

As preliminary verification of the interaction capacity between silica and chitosan networks, the pH of the precursor solutions and the mixtures were compared with the Point of Zero Charge point of silica (PZC = 2). At 21 °C, the pH of the chitosan precursor solution (4 wt% in acid acetic solution) is equal to 4.3 (pH-meter measurement). Comparatively, the pH of all the mixture solutions were determined to be in the same acid range (pH paper comparison), meaning that the pH of the solutions is always higher than silica PZC. In that conditions, silica particles are negatively charged due to predominance of Si-O groups, which gives them a hydrophilic character favorable to electrostatically interact with the $NH_3$ groups of chitosan.

## 3.2. Chemical structure of the films

The structure of the hybrid films was first studied by FTIR-ATR spectroscopy to evaluate the influence of the CS:TEOS ratio onto the network structure. Fig. 1 shows comparison between pure CS film and CS/SiO₂ with 90 wt% TEOS. Spectra of all other compositions are given in the Supporting Information (Fig. S1).

For pure chitosan film spectrum, bands associated with the vibration of primary amines bonds (–NH₂) around $1404 \text{ cm}^{-1}$ and secondary amines bonds (–NH–) around $1538 \text{ cm}^{-1}$ are present, as well as the residual amide absorption band around $1645 \text{ cm}^{-1}$ attributed to the incomplete deacetylation of chitosan. Other bands characteristic of C–H stretching mode of chitosan are also visible between ≈900–1100 $\text{cm}^{-1}$ and around 2877 $\text{cm}^{-1}$. Then, for all compositions incorporating TEOS, the characteristic bands of chitosan remain visible. For comparison, all the spectra were normalized on the C–H bond of chitosan at 2877 $\text{cm}^{-1}$. The incorporation of TEOS in the composition of the films is marked by the appearance of new bands around 1022 and 786 $\text{cm}^{-1}$ attributed to the vibration of siloxane bonds (respectively Si-O-Si asymmetric and symmetric stretching mode [50,51]), and around 950 $\text{cm}^{-1}$ associated with Si-OH. The presence of these signals shows that the hydrolysis of TEOS and the condensation of silica successfully occurred in these soft conditions of reaction. Moreover, a band around 1052 $\text{cm}^{-1}$ could be associated with Si-O-C symmetric stretching vibrations [52]. According to Bravo-Flores et al. [53] and Liu et al. [54], the existence of this band would confirm that chitosan intervenes in the condensation of Si-OH to form the Si-O-C bonds, limiting the self-condensation between Si-OH. At this stage, the FTIR signals are not enough clear to assume with evidence the presence of a covalent bond between the two networks because the frequency signal is located in the region of the strong band of the Si-O-Si asymmetric vibration, but also because this band could indicate that the

hydrolysis of TEOS is incomplete.

Also, while in the literature the peak around $786 \text{ cm}^{-1}$ is mostly attributed to the symmetric stretching mode vibration of silica, Ohishi et al. [55] and Larrieu et al. [35] have associated this band with Si-N vibration indicating a creation of a link between Si and N, i.e. a covalent bond between the silica and the chitosan, creating then a cross-linked networks.

Similar results are observed for the whole TEOS compositions (Fig. S1). By increasing TEOS amount, the band characteristic of the Si-O-Si bonds vibrations (around $1062 \text{ cm}^{-1}$ and $1019 \text{ cm}^{-1}$), and of the potential Si-O-C bond around $1062 \text{ cm}^{-1}$ are more and more intense, as well as the peak observed at $786 \text{ cm}^{-1}$. Conversely, the bands around $1404 \text{ cm}^{-1}$ and $1538 \text{ cm}^{-1}$ which are characteristic of the amine groups, are less and less intense while TEOS replaced chitosan in the composition.

The same analyses were performed on the films incorporating AZ dye to determine the impact of the pigment introduction during the reaction process on the final structure of the films. The CS/SiO₂ and CS/SiO₂/AZ films spectra with 90 wt% of TEOS are compared in Fig. 2A and Fig. 2B. For both compositions, the characteristic peaks of primary amines bonds (–NH₂) around $1404 \text{ cm}^{-1}$ and secondary amines bonds (–NH–) around $1538 \text{ cm}^{-1}$ of chitosan, the silica (Si-O-Si) vibrations (around $1022 \text{ cm}^{-1}$), and the peak at around $1052 \text{ cm}^{-1}$ that could correspond to a Si-O-C bond are observed, meaning that the introduction of alizarin in the composition of the hybrid films is not affecting the formation of the networks. As in the case of the films without alizarin, amine characteristic peaks are less intense in the film with 90 wt.%TEOS than for pure CS/SiO₂ film, due to the diminution of chitosan in the composition. Similar observations are obtained for the different CS:TEOS ratio confirming the network formation (Fig. S2A).

Fig. 2C gives the alizarin raw powder spectrum (black line) and shows the comparison between a pure chitosan film and films with 90 wt % TEOS with and without alizarin.

Among the characteristic bands of anthraquinone molecules, alizarin spectrum shows a broad band around $3400 \text{ cm}^{-1}$ characteristic of the hydroxy group OH. The band at $1662 \text{ cm}^{-1}$ is characteristic of the C=O carbonyl groups, and the bands at 1586 and 1450 $\text{cm}^{-1}$ correspond to the C=C of the aromatic rings, both of the anthraquinone molecules. First, it is worth noting that the characteristic peaks of alizarin are not observed on the different films containing the dye. This is probably due to the weak amount of pigment into the films ($4.10^{-4}$ wt%) that are overlapped with the other bands from chitosan and silica. In the case of the films containing 90 wt% TEOS with and without alizarin, the slight intensity differences in the range of 1200–500 $\text{cm}^{-1}$ are probably due to inhomogeneities of composition. These differences are observed for all the films containing 60 wt% TEOS and more (Fig. S2C).

Solid NMR spectroscopy was used in addition to ATR-FTIR to

<figure><img src="image_5.png" /><figcaption>Fig. 1. A) ATR-FTIR spectra of CS/SiO₂ hybrid films with 0 wt% and 90 wt% of TEOS. B) Zoom on –NH–, –NH₂, Si-O-C and Si-O-Si specific bands.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** ATR-FTIR spectra of A) CS/SiO₂/AZ hybrid films with 0 wt% and 90 wt% of TEOS, B) Zoom on -NH-, -NH₂, Si-O-C and Si-O-Si specific bands, and C) comparison between CS/SiO₂ hybrid films and CS/SiO₂/AZ hybrid films with 0 wt% and 90 wt% of TEOS.</figcaption></figure>

investigate the formation of bonds between the chitosan and silica networks and/or with alizarin. Fig. 3A shows the ¹³C and ²⁹Si NMR spectra of alizarin dye and CS/SiO₂ films containing 0, 50 and 90 wt% of TEOS without and with alizarin.

Whatever the composition of the films, similar spectra are obtained. The peaks observed are characteristic of chitosan with around 173 ppm a peak that corresponds to the -NHCOCH₃ function in correlation with the peaks between 25 and 102 ppm, characteristic of a saccharide moiety. The peak centered at 180 ppm is then attributed to the -COOH function of the residual acetic acid used as solvent for the solubilization of the chitosan. The similarity of the spectra of chitosan film without and with TEOS indicates the non-specific interaction/reaction between the two networks *via* Si-C bond. Concerning alizarin, after 15 h of accumulation, no aromatic signal characteristic of the dye or new chemical bond were detected on the films due to its weak concentration.

The ²⁹Si NMR spectra of the same films (Fig. 3B) show an important dilution of the silica network into the films, but the signals demonstrate the presence of Q sites around -110 (Q₄), -100 (Q₃) and -91 ppm (Q₂).

in similar proportions whatever the composition of the films (Fig. S3 and Table S3), indicating the presence of Si-O-C bonds and so confirming the observation of the Si-O-C FTIR band that could correspond to a covalent bonding between the two networks or the incomplete hydrolysis of TEOS. Nevertheless, no specific T sites peaks are observed around -80 (T₃), -70 (T₂) and -62 ppm (T₁) which could indicate the creation of Si-N or Si-C bonds between the chitosan and silica networks.

Thus, according to FTIR and NMR analyses, the structure of the CS/SiO₂ and CS/SiO₂/AZ films obtained by sol-gel route in soft conditions can be described either as semi-interpenetrated chitosan and silica networks linked by electrostatic interactions, or as interpenetrated networks if linked through Si-O-C covalent bonds as shown in Scheme 2.

**Films homogeneity.** SEM/EDS (Fig. 4) analyses were performed on the surfaces of pure CS and CS/SiO₂ films containing 50 and 90 wt% of TEOS to study their structure and homogeneity while increasing TEOS amount. On SEM images (Fig. 4a-c), the films' surfaces appeared similarly homogeneous with a small rugosity that seems to be slightly smoother for the film containing 90 wt% of TEOS. The homogeneity of

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** A) Solid state ¹³ C NMR spectra of alizarin and CS/SiO₂ films containing 0, 50 and 90 wt% of TEOS; B) Solid state ²⁹ Si NMR spectra of CS/SiO₂ films containing 50 and 90 wt% of TEOS.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Scheme 2.** Schematic representation of the interactions between chitosan and silica networks through A) electrostatic interactions between silica nanoparticles and chitosan, and B) covalent Si-O-C bonds.</figcaption></figure>

the films was confirmed by EDS chemical analyses. Indeed, spectra on Fig. 4d–f give evidence of the growing presence of silicon while increasing the amount of TEOS in the films, and Fig. 4g and Fig. 4h show their good dispersion/distribution through silicon mapping.

The films surfaces were also analyzed on AFM in adhesion mode (Fig. S4). Images show again a good homogeneity of the films without significant differences depending on their composition. No evidence of

nano or microdomains was observed by AFM or SEM.

### 3.3. Mechanical properties of films

The CS/SiO₂ hybrid films without and with AZ were studied through tensile tests. Fig. 5 shows photos of 100 % CS testing samples and CS/SiO₂ hybrid containing 90 wt% of TEOS. Photos of the testing samples of all other compositions are given in Fig. S5. The Young modulus (YM), the tensile strength (TS) and the elongation at break (EAB) parameters are reported in Fig. 6A, Fig. 6B, and Fig. 6C respectively. The tensile stress versus strain curves are given in Fig. S6.

It is worth noting that the films prepared with pure CS had a Young modulus around 1 450 MPa (Fig. 6A). The addition of TEOS in the composition of the hybrid films significantly increases the YM to reach about 4 000 MPa for the films containing 80 and 90 wt% TEOS. The tensile strength tends also to increase by addition of TEOS. Pure CS films show a TS of 46 MPa, while films with 80 and 90 wt% TEOS have a TS of 68 and 55 MPa, respectively, representing 48 and 20 % of improvement. The weaker TS observed for 60 and 90 wt% TEOS might be due to inhomogeneities in the CS/SiO₂ networks.

The increase in YM and TS of CS-based films incorporating silica is in accordance within the literature [56,57], showing that the soft

<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Photos of a) 100% CS testing samples and b) CS/SiO₂ hybrid containing 90 wt% of TEOS.</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>**Fig. 4.** SEM/EDS analyses of pure CS film surface (a, d), CS/SiO₂ containing 50 wt% of TEOS (b, e, g), and 90 wt% of TEOS (c, f, h). a), b) and c) are the SEM images. d), e) and f) are the EDS spectra. g) and h) correspond to the silicon EDS map.</figcaption></figure>
<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /><figcaption>Fig. 6. A) Young Modulus, B) Tensile strength and C) Elongation at break of CS/SiO₂ and CS/SiO₂/AZ hybrid films with 0 wt%, 50 wt%, 60 wt%, 70 wt%, 80 wt% and 90 wt% of TEOS.</figcaption></figure>

conditions used herein allow to create films with higher strength with a large extent of compositions.

Conversely, it is observed that the substitution of CS by TEOS significantly reduces EAB (Fig. 6C) from 35 mm/mm for pure CS films to 2 mm/mm for 10/90 wt% CS/TEOS films.

The increase in stiffness and tensile strength resistance by addition of silica into the films compositions, while decreasing their ductility, is attributed to the interpenetration of the chitosan and silica networks as shown by FTIR and ²⁹Si NMR analyses, and to the formation of hydrogen/electrostatic interactions [58]. Therefore, the amount of TEOS incorporated into the films will have to be adjusted in accordance with the targeted applications to reach the corresponding strength to flexibility balance.

Similar mechanical properties of hybrid films containing alizarin are observed. At this low concentration of dye, no significant differences are observed between the films without and with AZ. Slightly weaker

mechanical properties could be observed, nevertheless it can be considered in margin of error (less than 5 %). Young modulus increases from 1 406 MPa for CS/AZ to 3 202 MPa for CS/SiO₂/AZ films containing 90 wt% TEOS, representing an improvement of 128 %. An improvement of 42 % of the tensile strength is obtained by the substitution of chitosan with 80 wt% of TEOS for CS/SiO₂/AZ films. Finally, elongation at break measurements show a reduction from 33 mm/mm for CS/SiO₂/AZ films containing 50 wt% TEOS to 2 mm/mm for CS/SiO₂/AZ films containing 90 wt% TEOS.

If the addition of TEOS seems to drastically change the mechanical properties of the hybrid films, the presence of a very small amount of AZ does not impact the final properties of the colored hybrid films.

### 3.4. Films stability into aqueous media and swelling ratio

Stability and swelling tests were performed by immersing the films in MilliQ water (pH = 6.4) and in artificial sea water (pH = 8.2). These two different media were chosen to evaluate the influence of pH/salts on the solubility of the films. After 1 h of immersion in MilliQ water, pure chitosan films and films containing up to 60 wt% TEOS were dissolved while all others compositions (70 wt%, 80 wt% and 90 wt% TEOS, without and with alizarin) stayed stable until the end of the test (48 h). In artificial sea water, all the films stayed stable until the end of the test. These results show evidence that chitosan network is affected by the acid conditions. Amino groups of CS could be protonated in acid conditions leading to positively charge groups -NH³⁺. Electrostatic repulsion between these groups is then responsible for the network swelling until its dissolution [59]. It appeared also that the substitution of chitosan by more than 60 wt% of TEOS helped to prevent dissolution of the films in acid conditions by increasing the semi-interpenetration of the silica network with the macromolecular chains. This phenomenon could be also in accordance with the presence of covalent bonds between chitosan and silica networks that could increase through the addition of TEOS.

Swelling properties of the CS/SiO₂ hybrid films without and with alizarin were studied to measure the hydration of the chitosan/silica network. Fig. 7 shows swelling ratio (SR) measured in MilliQ water (Fig. 7A) and in artificial sea water (Fig. 7B), after 1 h of immersion in the media to reach the equilibrium.

In both conditions, SR depends on the TEOS content in the composition of the films. In MilliQ water, the SR ratio could not be measured for films containing less than 60 wt% TEOS due to their dissolution. For the other compositions, increasing the proportion of TEOS reduces the SR from about 500 % up to 50 % for films with 70 and 90 wt% TEOS, respectively (Fig. 7A). In artificial sea water (pH = 8.2), a maximum SR of about 155 % is observed for pure chitosan films and up to 50 wt% TEOS in the composition (Fig. 7B). It then decreases with the augmentation of TEOS ratio to stabilized around 70 to 50 % for films containing more than 80 wt% of TEOS. Liu et al. [60] had reported that the swelling properties of chitosan based films containing nanosilica spheres were decreased with the increase of nanosilica. Indeed, as chitosan films form a dense network with dispersed nanosilica, it decreases the number of hydrophilic groups. They also reported the same tendency for the pH effect on the swelling properties of CS/SiO₂ hybrid films. They observed that the swelling ratio of their films at pH 2.6 was rather higher than films at pH 7.6 whatever the amount of silica [60].

In both conditions of pH, results were similar for films containing alizarin dye meaning that this last one does not affect the cohesion of the films at this low concentration.

### 3.5. Wettability measurement

Contact angle measurements were performed both with MilliQ water and artificial sea water to minimize the influence of the pH on the results due to water absorption. Fig. 8A presents values obtained after deposition of MilliQ water drops on the surface of the films without and with
<figure><img src="image_14.png" /><figcaption>Fig. 7. Swelling ratio of CS/SiO₂ and CS/SiO₂/AZ hybrid films with 0 wt%, 50 wt%, 60 wt%, 70 wt%, 80 wt% and 90 wt% of TEOS. In MilliQ water (A) and artificial sea water (B).</figcaption></figure>

alizarin.

Results are similar in both cases meaning that the presence of alizarin does not modify the wettability of the films. Pure chitosan films show a contact angle of 76° and 78° without and with alizarin respectively. Hydrophily of the film surfaces seems to slightly decrease while increasing the amount of silica, to reach 83° for the films containing 70 wt% TEOS. Above 70 wt% TEOS, the contact angle decreases to equal the value of pure chitosan films. The same tendency was observed for the tests with artificial water (Fig. 8B). This could be due to a competition between the wettability of the films and a fast diffusion of the water drop into the film.

Even though we expected to obtain the reinforcement of hydrophily of the films due to the augmentation of silica in the composition, the difference in contact angle values was not significant. That could be explained by the presence of chitosan at the surface of the hybrid films whatever their composition [35].

### 3.6. Optical properties of the films

The optical properties of the hybrid films were measured both in transmittance and in absorbance spectroscopy in the range of 250–800 nm. The films were deposited onto glass plates to obtain a flat surface. Fig. 9A gives a comparison in transmittance between CS and CS/SiO₂ hybrid films containing 90 wt% TEOS, with and without alizarin. Spectra of all the other compositions are given in the Supporting Information (Fig. S7). Fig. 9B shows the corresponding absorbance spectra. Relative values could not be compared due to differences in the films' thicknesses (Table S4).

Nevertheless, it was observed that the different films present a good transparency to visible light between 800 and 500 nm. At smaller wavelengths, chitosan films present an important absorbance, particularly around 310 nm. This loss in transparency is much less pronounced for the film with 90 wt% TEOS, and is attributed to the decrease in

chitosan amount for this composition. For the films containing alizarin, no significant differences are observed in the spectra except in the region 500–550 nm where a slight broad absorbance peak is visible and attributed to the orangish color of the pigment.

### 3.7. pH-sensitiveness of CS/SiO₂/AZ hybrid films

An interesting property of alizarin dye is its important pH sensitivity resulting in a wide range of colors. This can be advantageous for abnormal acid or basic conditions detection for food packaging, by example. The influence of the pH conditions on the chemical structure and the color of alizarin is presented in Fig. 10.

At pH lower than 5.2, AZ is fully protonated and the solution appears to be orange-yellow with a UV absorption spectrum centered at 460 nm. Between pH 8 and pH 10, AZ solutions are observed to be orange-red. This protonation/deprotonation process is reversible in this range of pH. The absorption peak at 460 nm is shifted to 515–530 nm and a second absorption peak is observed at 330 nm. The hydroxyl group in β position is deprotonated. At pH higher than 10, the second hydroxyl group, i.e. in α position, is then also deprotonated leading to a purple solution. The absorption spectra show peaks at 530 nm, 568 nm and 608 nm. The peak at 330 nm tends to be shifted to wavelengths lower than 300 nm while pH value increases.

The pH-sensitiveness of AZ into films was evaluated by UV-vis spectroscopy both under acid/basic atmospheres (Fig. 11) and by drop deposition of an acid/basic solutions (Fig. 12).

Fig. 11 shows the results obtained on CS/AZ and CS/SiO₂/AZ (90 wt % TEOS) films, either exposed first to an acid atmosphere (Fig. 11A,B) or first to a basic atmosphere (Fig. 11C,D). For the test performed first under acid conditions, it can be seen a clear change in color from orange to purple after 24 h under basic atmosphere (pictures on Fig. 11A,B). These results indicate that alizarin is sensitive to its environment while incorporated into the films. This pH-sensitiveness is also visible on the

<figure><img src="image_15.png" /><figcaption>Fig. 8. Contact angle measurements for CS/SiO₂ and CS/SiO₂/AZ hybrid films with 0 wt%, 50 wt%, 60 wt%, 70 wt%, 80 wt% and 90 wt% of TEOS. A) Using MilliQ water, and B) using artificial sea water.</figcaption></figure>
<figure><img src="image_16.png" /><figcaption>Fig. 9. UV-light spectra of CS and CS/SiO₂ hybrid films with 90 wt% of TEOS with and without alizarin in A) transmittance, and B) absorbance mode.</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>Fig. 10. A) Chemical forms of alizarin dye at different pH, B) Photos of alizarin at different pH in buffer solutions (2.10⁻⁴ M) and C) UV-vis spectra of alizarin in buffer solutions at different pH.</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>Fig. 11. UV-vis spectra in absorbance of A, C) CS/AZ films and B, D) CS/SiO₂/AZ films containing 90 wt% TEOS, under acid and basic atmosphere. Figures A, B) refer to the test first performed under acid then basic atmosphere. C and D) refer to the test first performed under basic then acid atmosphere.</figcaption></figure>
<figure><img src="image_19.png" /><figcaption>Fig. 12. UV-vis spectra in absorbance of A) CS/SiO₂ films and B) CS/SiO₂/AZ films with 90 wt% TEOS, under acid and basic projections.</figcaption></figure>

UV-vis spectra with a slight increase of the bands around 250 nm, 300 nm and 550 nm. This behavior goes in favor of an increase of the pH around AZ as presented previously (Fig. 10). Moreover, it also seems to indicate that in the reference films, alizarin is in an acid environment lower than pH 5 which correspond to the synthetic conditions (chitosan powder was solubilized in a 2 wt% acid acetic solution). Similar results were obtained for the other composition tested (CS/SiO₂/AZ with 70wt. %TEOS). When the films are first exposed to a basic environment (Fig. 11C,D), the spectra showed again an increase in the intensity of the characteristic bands at 250 nm, 300 nm and 550 nm and the films become purple. Nevertheless, no reversibility of the pH-sensing was observed. Similar results were obtained for all other compositions tested (CS/SiO₂/AZ with 50wt.%TEOS and CS/SiO₂/AZ with 70wt.%TEOS) (Fig. S8).

The reversibility of the pH-sensing of CS/SiO₂/AZ films was then studied by successive depositions of acid and basic drops on the films surface. First, some HCl solution drops @ pH 5 were deposited on the samples surface and the corresponding spectra were register after 1 min of exposure (sample drop pH 5-1). Then, multiple KOH solution drops @ pH 10 were deposited on the same area (enough to wash the acid solution used before) and the corresponding spectrum was registered (sample drop pH 10-2). Results are presented on Fig. 12A and Fig. 12B, i.e. respectively CS/AZ and CS/SiO₂/AZ 90wt.%TEOS films.

Similar behaviors are observed for the tests 1 and 2, respectively under acid then basic conditions compared to previous acid/basic atmospheres (Fig. 11A,B). Indeed, an intensity increase of the characteristic bands is visible associated with a bathochromic effect, i.e. slight shift to the higher wavelengths. Analogous results are obtained for the other films compositions (Fig. S9). Nevertheless, due to the films swelling, we observed a pigment desorption/release (see insert Fig. 12B) decreasing drastically the dye content and making reversibility tests non-significant (tests 3, 4 and 5, respectively acid pH 5, basic pH 12, acid pH5). Here in a more sustainable approach, it is worth noting that the alizarin dye can be chemically modified to be grafted on macromolecules [61] to avoid leaching and so a one-shot pH-sensor. Nevertheless, this last point can be turned in a positive way to develop one-shot pH-sensitive film for food protection to avoid fraud or reconditioning.

## 4. Conclusion

Mimicking the biosilicification occurring in the diatom frustule to obtain hybrid organic/inorganic films was possible using a sustainable approach with chitosan biopolymer to polycondense in-situ silica from tetraethoxysilane (TEOS) precursor via a simple sol-gel aqueous route in soft conditions. We showed that:

1. self-supported cohesive hybrid silica films can be obtained varying the TEOS amount from 0 to 90 wt%;

2. it was possible to design the mechanical and optical properties of the films managing the TEOS content. Nevertheless, as chitosan is soluble into acid media, a minimum amount of 70 wt% of TEOS

was necessary to keep a cohesive film in MilliQ water while the films are stable in saline solution mimicking Atlantic Ocean water.

3. it is possible to incorporate a pH-sensitive organic dye without affecting the films properties.

4. at this stage, the use of alizarin showed that the dye is still sensitive to its environmental conditions while incorporated in the films whatever the composition.

If only one-shot reversibility of the pH-sensitiveness of the colored films was demonstrated due to the release of the dye, for future improvements, alizarin dye could be grafted to develop sustainable pH-sensing films [61-63] for multiple uses as food packaging to follow the expiry date or food degradation, especially.

## CRediT authorship contribution statement

**Sandra Castanié:** Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Data curation. **Laurent Billon:** Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

## Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Laurent Billon reports financial support was provided by Rectorat de Bordeaux. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper].

## Acknowledgment

The authors thank E2S UPPA and Rectorat Nouvelle Aquitaine for the financial support of the "DSG2 Biomimétisme" project, and the S.C. fellowship especially.

## Appendix A. Supplementary material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.matdes.2024.113571.

## Data availability

No data was used for the research described in the article.

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