# Monitoring of lipid oxidation using a fluorescent sol-gel functionalized material

Laura Carballido, Virginie Guerlet, Elias Bou-Maroun, Thomas Karbowiak

► To cite this version:

Laura Carballido, Virginie Guerlet, Elias Bou-Maroun, Thomas Karbowiak. Monitoring of lipid oxidation using a fluorescent sol-gel functionalized material. Sensing and Bio-Sensing Research, 2025, 49, pp.100848. 10.1016/j.sbsr.2025.100848. hal-05244413

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# Monitoring of lipid oxidation using a fluorescent sol-gel functionalized material

Laura Carballido, Virginie Guerlet, Elias Bou-Maroun *, Thomas Karbowiak

Université Bourgogne Europe, Institut Agro, INRAE, UMR PAM, F-21000 Dijon, France

## ARTICLE INFO

**Keywords:**

Sol-gel

Fluorescence

Carbonyls

Lipid oxidation

Sensor

## ABSTRACT

The food industry lacks rapid, easy-to-use and non-toxic analytical methods to assess the oxidation level of lipid-rich products. A new fluorescent sol-gel sensing material was developed for the fast and simple monitoring of carbonyls secondary products of lipid oxidation. This paper defines the sensor's range of applicability, specifically examining the impact of the nature and structure of the detected carbonyl compounds, as well as the effects of pH and the nature of the food-simulating medium on its response. The material exhibited high fluorescence intensity, which was quenched upon interaction with carbonyls. The size and hydrophobicity of the analyte (propanal, hexanal, nonanal, 1-penten-3-one and 1-octen-3-one) impacted the kinetics and the percentage of fluorescence quenching as well as the limit of detection and the saturation limit of the sensor. Additionally, the solvent nature had a significant impact on fluorescence: fluorescence intensity was higher in ethanol compared to the more polar ethanol/water (10/90, v/v) mixture used as a food simulant. Finally, the optimal pH range to operate the sensor was determined to be from 7 to 9. This study thus validated the feasibility of using this new sensor for the fast (less than 5 min) and simple (no sample preparation) monitoring of food oxidation level without using any toxic reagent compared to conventional methods. The sensor enables global carbonyl content determination within an operative pH range and a range of detection that would cover the entire shelf-life of lipid-rich food products.

## 1. Introduction

Lipids are essential nutrients in human diet. They are made of polyunsaturated fatty acids such as ω3, which are known to play a crucial role in reducing the risk of cardiovascular disease or in regulating inflammatory reactions [1,2]. However, lipids are very sensitive to oxidation, which is a natural chemical degradation process. Initiated either by heat, light, sensitizers or metals, the radical process involved is unstoppable [3]. During lipid oxidation, many different reaction products are generated. The first ones, so-called "primary products", are mainly hydroperoxides [3,4]. They are further transformed into "secondary products". Among them, aldehydes, ketones, alcohols and carboxylic acids are the most released molecules [4]. Many of them are volatiles and responsible for the unpleasant rancid odor, characteristic of lipid oxidation [5].

Since it is difficult to avoid lipid oxidation, its monitoring is of high importance. Many common and standard methods for the determination of the lipid oxidation level of a food product already exist. The monitoring of primary products relies on the measurement of the peroxide

value (PV) [6] or on the determination of the amount of conjugated dienes (CD233) [7]. Secondary products are usually monitored by the estimation of the p-anisidine value [8], by the thiobarbituric acid reactive substances (TBARS) assay [9], by chromatographic methods or even by sensory evaluation. Although these methods have been used for decades, they still have some limitations, as they often require highly trained personal, advanced laboratory skills, significant time and expensive equipment. The primary limitation of the TBARS assay is its long processing time (around 3 h). The p-anisidine value method involves the use of highly toxic reagents, particularly p-anisidine, which is classified as carcinogenic (H350). Chromatography requires expensive, complex equipment and must be operated by highly trained personnel. Sensory evaluation is also an expensive and complex method as it depends on trained panelists to smell each sample.

Fluorescence has been used to monitor lipid oxidation of food products, but not through the use of fluorescent sensing materials. Firstly, fluorescence has been used to detect fluorescent compounds formed during the oxidation process, such as Schiff base products. These arise from the reaction of aldehydes generated by lipid oxidation with

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: elias.bou-maroun@institut-agro.fr (E. Bou-Maroun), thomas.karbowiak@institut-agro.fr (T. Karbowiak).</footnote>
fluorescent amino acids like tryptophan, tyrosine of phenylalanine present in food. A limitation of this approach is the need to anticipate which specific compounds will be present in order to appropriately adjust excitation and emission wavelengths. Furthermore, this measurement is typically taken from the food's surface, which may not accurately reflect the oxidation level of the entire product [10]. Secondly, the TBARS assay, commonly performed using a UV-visible spectrophotometer, could also be adapted to fluorescence detection. This assay is based on the formation of a compound from the reaction of malonaldehyde with thiobarbituric acid, which exhibits both absorbance and fluorescence [11]. Thirdly, chromatographic techniques could be coupled with fluorescence detectors to monitor carbonyls compounds produced during lipid oxidation. This approach requires a derivatization step, typically involving 2,4-dinitrophenylhydrazine, which reacts with carbonyl compounds to form fluorescent dinitrophenylhydrazone derivatives [12].

In the present work, the performance of a user-friendly sensor intended to monitor lipid oxidation was thoroughly evaluated. Based on the fluorescence of an aniline derivative and synthesized by a cost-effective and straightforward sol-gel process, a sensing material was developed to track secondary products of lipid oxidation, especially carbonyl compounds. Aniline was chosen to act as fluorescent probe. Its primary amine function was intended to chemically react with carbonyl compounds. The synthesized material exhibited high fluorescence intensity, which was quenched upon interaction with carbonyls. Details about the synthesis and characterization of this material can be found in previous work [13,14]. As this sensor was designed to indicate the global content of carbonyls, the impact of the nature and the structure of the analyte as well as the influence of the solvent polarity and its pH on the fluorescence property of the material were assessed. More precisely, the kinetics of the fluorescence decrease and the percentage of fluorescence quenching of the material by different carbonyls (namely propanal, hexanal, nonanal, 1-penten-3-one and 1-octen-3-one) as well as the limit of detection and saturation limit for each carbonyl were compared in ethanol and in an ethanol/water (10/90, v/v) mixture, used as food simulant medium. These specific carbonyl compounds were identified as the main representatives of secondary products released during lipid oxidation of various food matrices (meat [15,16], fish [17–20], fish oils [21] and vegetable oils [4,22,23]). They originate from different substrates: propanal [24] and 1-penten-3-one [25] are derived from ω3-linolenic acid; hexanal [26] and 1-octen-3-one [25] from ω6-linoleic acid; and nonanal [27,28] is an oxidation product of ω9-olenic acid). Moreover, the impact of interfering compounds (n-pentane, n-hexane, n-octane) on the fluorescence of the material was assessed. The pH range in which the sensor was operational was finally determined.

## 2. Materials and methods

### 2.1. Chemicals

4-trimethoxysilylaniline (4-TMSA, 97 %, CAS number 33976–43–1) was obtained from abcr, Germany. Tetraethylorthosilicate (TEOS, 99 %, CAS number 78–10–4), ammonium hydroxide (NH₄OH, 30 %, CAS number 1336–21–6), hydrochloric acid (HCl, CAS number 7647–01–0), sodium hydroxide (NaOH, CAS number 1310–73–2), 1-octen-3-one (96 %, CAS number 4312–99–6), n-pentane (99 %, CAS number 109–66–0), n-hexane (97 %, CAS number 110–54–3), 1-penten-3-one (97 % CAS number 1629–58–9), formic acid (98 %, CAS number 64–18–6), ammonium hydroxide solution (28–30 %, CAS number 1336–21–6) and ethanol for UV-spectroscopy Uvasol® (EtOH, 99.9 %, CAS number 64–17–5) were purchased from Sigma Aldrich, France. n-octane (99 %, CAS number 111–65–9) was supplied by Merck, Germany. Propanal (99 %, CAS number 123–38–6) was obtained from ACROS Organics™. Hexanal (98 %, CAS number 66–25–1) was purchased from Alfa Aesar, France. Nonanal (97 %, CAS number 124–19–6) was bought from Thermo Scientific Chemicals. Deionized water was used, obtained from

a MilliQ RG, Millipore apparatus.

### 2.2. Fluorescence experiments

#### 2.2.1. General procedure

A suspension of the sol-gel material in ethanol or in an ethanol/water mixture (50/50, v/v or 10/90, v/v) was realized. After 10 s of stirring, the fluorescence emission spectrum of the material in suspension was recorded. A carbonyl solution in ethanol at a given concentration (either propanal, hexanal, nonanal, 1-penten-3-one or 1-octen-3-one) was added to the dispersing phase and left in contact with the material in suspension for a given time before the fluorescence emission spectrum of the material was recorded once again. Both influence of contact time and concentration of carbonyl were studied. A spectrofluorometer Fluoromax4® (Horiba) was used with the software Fluorescence® and a SUPRASIL® quartz cell (Hellma) of 10 mm light path. Excitation wavelength was 290 ± 2 nm. The fluorescence emission spectrum was recorded between 300 nm and 500 nm, using a 2 nm bandpass and a step interval of 1 nm. The temperature in the room was kept constant at 22 °C during the experiment. Each experiment was performed in triplicate.

#### 2.2.2. Kinetics of decrease of the fluorescence intensity of the material after interaction with different types of carbonyls

A suspension of 10 mg of material in 2 mL of ethanol was stirred for 10 s and the fluorescence emission spectrum of the material was recorded. Then, 500 µL of a solution of carbonyl (either propanal, hexanal, nonanal, 1-penten-3-one or 1-octen-3-one) in ethanol was added to obtain a concentration in the measurement cell of 9.98 × 10⁻³ mol·L⁻¹. The cell was then continuously agitated at 80 rpm, and the fluorescence emission spectrum of the material was recorded after 30 s, 5 min, 10 min, 15 min, 20 min, 25 min and 30 min. The fluorescence intensity of the material at 337 nm was compared at each time.

#### 2.2.3. Percentage of fluorescence quenching

The percentage of fluorescence quenching, Q, was determined as follows:

$$Q = \frac{I_0 - I_{eq}}{I_0} \times 100 \qquad (1)$$

with $I_0$ the intensity of fluorescence at 337 nm of the material in ethanol before addition of carbonyl and $I_{eq}$ its intensity after exposure to carbonyl for 20 min.

#### 2.2.4. Influence of the concentration of the analyte on the fluorescence of the material

A suspension of 10 mg of the material in 1.8 mL of ethanol was placed in a quartz cell. After 10 s of stirring, the fluorescence spectrum of the material was recorded. A known amount of carbonyl in ethanol was periodically added to increase the carbonyl concentration in the dispersing phase from 0 mol·L⁻¹ to 0.54 mol·L⁻¹. The solution was agitated at 80 rpm for 10 min to reach the equilibrium after each addition and the fluorescence emission spectrum of the material was recorded.

To test the response of the sensor in the presence of interfering compounds, the influence of an increasing concentration of either n-pentane, n-hexane or n-octane on the fluorescence of the material was also assessed. The same experiment as previously described was performed replacing the carbonyls by their alkane structural analogue.

#### 2.2.5. Impact of the polarity of the solvent on the fluorescence of the material

After 10 s of stirring, the fluorescence emission spectrum of a 10 mg suspension of the material in ethanol, ethanol/water (50/50, v/v) mixture, ethanol/water (10/90, v/v) mixture and water were recorded. Each spectrum was recorded three times. The kinetics of decrease of
fluorescence intensity, the percentage of fluorescence quenching and the influence of the concentration of the analyte on the fluorescence of the material were determined, as described in 2.2.2, 2.2.3 and 2.2.4.

### 2.2.6. Impact of the pH of the dispersing phase on the fluorescence of the material

The pH of ethanol/water (10/90, v/v) mixtures was adjusted using HCl or NaOH in order to obtain solutions at pH of 1.4, 3.6, 7.6, 9.3 and 12.8. A 5 mg suspension of material was realized in 2 mL of each of these solutions. After 24 h of stirring at 50 rpm, the fluorescence emission spectrum of the material was recorded. Then, 500 µL of a solution of 1-octen-3-one in ethanol was added to obtain a concentration in the measurement cell of $2.3 \times 10^{-3} \text{ mol} \cdot \text{L}^{-1}$. The solution was agitated at 80 rpm for 10 min in order to reach the equilibrium and then, the fluorescence emission spectrum of the material was recorded. The measurements were performed in triplicate for each pH.

### 2.3. Structure determination of 1-PENTEN-3-one by HPLC-MS

Results obtained with 1-penten-3-one were unexpected. The hypothesis that this molecule could eventually dimerize was suggested. The structure of the conjugated ketones studied in this work was thus investigated by HPLC-MS. Solutions of 1-penten-3-one and 1-octen-3-one in acetonitrile at two concentrations ($5 \times 10^{-4} \text{ mol} \cdot \text{L}^{-1}$ and $5 \times 10^{-3} \text{ mol} \cdot \text{L}^{-1}$) were filtrated and outgased before injection in a 2695 liquid chromatograph (Waters) equipped with a C-18 column ($4.6 \times 50$ mm internal diameter, 3.5 µm thickness, XBridge™) and an Acquy QDa mass spectrometer detector (Waters). The mass spectrometer was scanned in the 50–1200 m/z range. Analytical conditions were as follows: 0.8 kV capillary voltage, 30 V cone voltage, 120 °C source block temperature, and the nitrogen flow was kept at 1200 L·h$^{-1}$. The compounds were analyzed using electrospray ionization (positive or negative modes). The separation was achieved using either acidic or basic conditions at a flow rate of $0.6 \text{ mL} \cdot \text{min}^{-1}$. The sample injection volume was 10 µL for each analysis. The temperature of the column and the autosampler was 25 °C. The mobile phase was composed of a mixture of two solvents. For positive ion mode, solvent A was a formic acid/water mixture (0.1/99.9, v/v) and solvent B was a formic acid/acetonitrile mixture (0.1/99.9, v/v). For negative ion mode, formic acid was replaced by ammonium hydroxide. The following gradient of solvent A and B was used for the separation: 10 % of B was maintained for 2 min and the percentage of solvent B was increased up to 90 % in 5 min. 90 % of B was maintained for 2 min before being decreased to 10 % in 2 min and maintained at 10 % for 2 more min. Acquired data were analyzed with the MassLynx® software, version 4.1.

### 2.4. Statistics

Statistical analysis (ANOVA with Tukey's post hoc analysis) was performed using XLSTAT (Addinsoft) software. Significance was established at *p* < 0.05.

## 3. Results and discussion

### 3.1. Influence of the ANALYTE chain length on the fluorescence property of the material

Propanal, hexanal and nonanal were selected in order to study the influence of the chain length of the aldehyde on the fluorescence property of the material. These three aldehydes are produced by the oxidation of ω3, ω6 and ω9 fatty acids, respectively [27,28]. Besides, only a few mono-ketones released during lipid oxidation are aliphatic. In order to have an example of ketones coming from the oxidation of different types of fatty acids and thus be able to compare them, two conjugated ketones were also chosen. 1-penten-3-one and 1-octen-3-one are also good trackers of ω3 and ω6 fatty acids degradation, respectively

[25] (see Fig. 1c). The experiments were first conducted in ethanol to assess the effect of the nature of the analyte, and particularly the effect of the chain length on the fluorescence property of the material.

### 3.1.1. Influence of the nature of the analyte on the kinetics of fluorescence decrease of the material

Fig. 2a and b depict the kinetics of the decrease of the fluorescence intensity of the material after interaction with different analytes. For all of the tested carbonyls, the equilibrium was reached in less than 5 min. The rapid response of the sensor represents a significant advantage for potential applications, as it offers a faster alternative to traditional methods typically used to monitor secondary products of lipid oxidation. From the chosen representation of $I_t/I_{eq}$ in Fig. 2a and b, it is easy to determine for which analyte the equilibrium was reached the fastest. The fluorescence of the material was quenched more rapidly with the ketones compared to the aldehydes. Moreover, the longer the hydrocarbon chain was, the faster was the quenching. Molecules which quenched the fluorescence faster ranked as follows: for aldehydes, nonanal (logP: 3.3) > hexanal (logP: 1.8) > propanal (logP: 0.6) and for ketones, 1-octen-3-one (logP: 2.4) > 1-penten-3-one (logP: 1.0). Therefore, it seemed that the more hydrophobic the molecules were, the faster they interacted with hydrophobic anilines groups of the material, resulting in rapid fluorescence quenching.

### 3.1.2. Influence of the nature of the analyte on the fluorescence quenching of the material

Fig. 2c and d display the percentage of fluorescence quenching upon interaction of the material with each target. It was observed that the shorter the molecule was, the more important the fluorescence decrease was. For example, propanal was responsible for 50 % of fluorescence quenching while nonanal was only responsible for 43 % of fluorescence extinction at the same concentration ($9.98 \times 10^{-3} \text{ mol} \cdot \text{L}^{-1}$). Among ketones, the molecular size impacted the percentage of fluorescence quenching in a similar manner to aldehydes. 1-penten-3-one was responsible for 57 % of fluorescence loss while 1-octen-3-one quenched the fluorescence only to the 40 % extent. Such a ranking of the molecules responsible for the most important quenching was obtained: for aldehydes, propanal > hexanal > nonanal, and for ketones, 1-penten-3-one > 1-octen-3-one. This could be explained by the fact that smaller molecules are more likely to easily access to more aniline sites and especially those that are entrapped within the bulk polymeric structure of the material. Smaller molecules could thus quench more aniline cores than molecules with a longer aliphatic chain for a given analyte concentration.

### 3.1.3. Influence of the nature of the analyte on the limit of detection and saturation limit of the sensor

The limit of detection (LOD) and the saturation limit of the material were determined thanks to the plot of the percentage of fluorescence quenching of the material according to the analyte concentration (Fig. 3a and b). In its application as a sensor for carbonyl monitoring, the LOD has to be as low as possible. The LOD values fall within the range typically achieved by the TBARS assay [29] and sensory evaluation [30], although they are higher than those expected from chromatographic methods [31]. However, unlike chromatographic techniques, which target the detection of individual compounds, the proposed sensor is non-selective and enables the detection of the overall content of carbonyl compounds. The saturation limit is also an important parameter to consider, as it determines the concentration range in which the sensor can operate. Actually, in order to be able to monitor the dynamic shelf-life of a product, the fluorescence extinction must occur above a given carbonyl concentration, corresponding to the spoilage threshold of the food. Saturation limit should thus be as high as possible. Fig. 3c and d display the limit of detection and saturation limit of the material for the different carbonyls tested. It was noteworthy that for aldehydes, the LOD increased, and the saturation limit decreased with the chain
<figure><img src="image_3.png" /></figure>

<table><thead><tr><th></th><th>Typical oxidation product of<br>ω3 fatty acids</th><th>Typical oxidation product of<br>ω6 fatty acids</th><th>Typical oxidation product of<br>ω9 fatty acids</th></tr></thead><tbody><tr><th>Aldehydes</th><td>Propanal</td><td>Hexanal</td><td>Nonanal</td></tr><tr><th>Ketones</th><td>1-penten-3-one</td><td>1-octen-3-one</td><td></td></tr></tbody></table>

**Fig. 1.** Structure of the sensing material before and after interaction with the carbonyl compounds. a. Structure of the synthesized sol-gel fluorescent sensing material. m and n are the molar ratio of functional monomer (4-trimethoxysilylaniline) and cross-linker (tetraethyl orthosilicate) used for the synthesis of the functionalized sol-gel material, respectively. b. Structure of the material after interaction with carbonyl compounds either by 1) chemical reaction between the amino group of the sol-gel sensing material and the carbonyl compound with formation of an imine 2) hydrophobic interactions between the hydrocarbon chain of the carbonyl compound and the aromatic ring of aniline 3) hydrogen bonds between the carbonyl group and uncondensed silanols. R and R' are alkyl groups. c. Structure of the different carbonyl compounds tested.

length. Among the three aldehydes tested, propanal thus exhibited the lowest LOD and the highest saturation limit. The molecular size and the hydrophobicity of the molecule could explain such a ranking in LOD. As previously suggested, smaller molecules could probably more easily enter inside the porosity of the material. Thus, they have more opportunities to interact with aniline groups entrapped within the bulk structure of the material and to give rise to fluorescence quenching. As the material displayed both micropores (from 0.5 to 1 nm) and mesopores (from 2 to 5 nm) [13], low concentrations of short-chain molecules are more likely to be detected by fluorescence quenching. The accessibility to quenching sites seemed also linked to the hydrophobicity of the molecule. The material was composed of 80 % hydrophilic mineral fraction and 20 % hydrophobic organic aniline [13]. As a consequence, longer molecules, but also more hydrophobic due to their longer aliphatic chain, may have restricted accessibility to the inner structure of the material. In order to better explain the differences observed for the saturation limit, the fluorescence quenching mechanisms need to be referred to. Upon the reaction of an aniline group with a carbonyl, an imine molecule is formed (see Fig. 1b). The fluorescence quenching of the material can first rely on intramolecular motions of the aliphatic part of the aldimine or the ketimine responsible for non-radiative relaxation [32–34]. It can be assumed that the longer the aliphatic chain is, the more energy will be dissipated by the motion of the hydrocarbon chain upon formation of the imine and thus the more the fluorescence will be quenched. Therefore, carbonyls with longer hydrocarbon chain will

quench the fluorescence to a larger extent at a given concentration. This could explain why the saturation limit was reached at lower concentrations with longer molecules.

Dealing with ketones, 1-octen-3-one exhibited a smaller LOD compared to 1-penten-3-one, although they both displayed similar saturation limits. Contrarily to aldehydes, the shortest ketone had the highest LOD. Such difference could be explained by the potential degradation of 1-penten-3-one. HPLC-MS analysis of the 1-penten-3-one solution revealed the presence of 4-methyl-1-nonene-3,7-dione, resulting from the dimerization of 1-penten-3-one [35] as well as 1-(6-ethyl-3,4-dihydro2(2H)-pyranyl)-1-propanone and 4-hydroxy-3,8-decanedione, being both products of the Diels-Alder reaction [35]. The molecular size and hydrophobicity of these molecules strongly differ from the properties of 1-penten-3-one, which could explain the inconsistency of the results obtained with the ketones.

### 3.1.4. Influence of interfering compounds on the fluorescence property of the material

The impact of the presence of interfering compounds on the fluorescence property of the material was also investigated. n-pentane, n-hexane and n-octane were chosen as alkane structural analogues of the different carbonyls tested. It was noteworthy that n-pentane did not affect the fluorescence emission spectrum of the material at all. However, n-hexane and n-octane were both responsible for 30 % of fluorescence quenching. Fig. 4a compares the influence of the concentration of
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Kinetics of the fluorescence decrease of the material after interaction with the selected carbonyls and corresponding percentage of fluorescence quenching at equilibrium. **a, b.** Kinetics of the fluorescence decrease of the material after interaction with aldehydes (a) and ketones (b). The intensity of fluorescence of the material in suspension in a solution of each carbonyl in ethanol (concentration: 9.98 × 10⁻³ mol·L⁻¹) was followed over time. *I*<sub>t</sub> corresponds to the fluorescence intensity of 10 mg of the material at 337 nm at a given time and *I*<sub>eq</sub> represents the fluorescence intensity of the material at 337 nm at equilibrium (considered at 30 min). Error bars represent the standard deviations obtained from three replicates. Solid lines are guides for the eyes. **c, d.** Percentage of fluorescence quenching of the material by aldehydes (c) and ketones (d) for the same carbonyl concentration (9.98 × 10⁻³ mol·L⁻¹) after 20 min of stirring of the material in suspension in each carbonyl solution in ethanol. Error bars represent the standard deviations obtained from three replicates. Statistical comparisons were performed using one-way ANOVA tests. Significantly different values (p < 0.05) are indicated with different number of stars.</figcaption></figure>

hexanal and *n*-hexane on the fluorescence of the material. Hexanal was able to quench 100 % of the fluorescence of the material, while a plateau at 30 % was reached with *n*-hexane. Only physical interactions such as hydrophobic interactions could explain the quenching of the fluorescence of the material by the alkane (see Fig. 1b). It could be deduced that several quenching mechanisms are involved when the material interacts with hexanal, and that some of them are related to physical interactions. Fig. 4b compares the kinetics of fluorescence quenching either induced by hexanal or by *n*-hexane. It was observed that the equilibrium was reached faster for *n*-hexane compared to hexanal. This is in line with previous results revealing that the more hydrophobic the molecules were, the faster the equilibrium was reached. For sensor applications, potential quenching by interfering compounds must be considered by incorporating a sufficient amount of sol-gel material to compensate for fluorescence losses caused by these compounds. This will also be addressed through prior calibration of the sensor for each specific matrix, particularly if such structural analogues are present in the sample (which is generally not the case for foods).

## 3.2. Influence of the solvent on the fluorescence property of the material

### 3.2.1. Influence of the polarity of the solvent on the fluorescence of the material

The impact of the nature of the solvent on the fluorescence property of the material was assessed by analyzing the fluorescence emission spectrum of the material in suspension in four different solvents of increasing polarity: ethanol, ethanol/water mixture (50/50, v/v),

ethanol/water mixture (10/90, v/v) and water (see Fig. 5a). In ethanol, the material was 1.7 times more fluorescent than in water. In mixtures of ethanol and water the fluorescence intensity decreased when the water content increased. Thus, the fluorescence intensity of the material decreased when the polarity of the solvent increased. Moreover, a slight bathochromic shift of the maximum emission wavelength of the material was noticeable when the solvent polarity increased: $\lambda_{max}$ (ethanol) = 338 nm > $\lambda_{max}$ (ethanol/water, 50/50, v/v) = 339 nm > $\lambda_{max}$ (water) = 340 nm. It is known that changing the solvent may impact the fluorescence maximum wavelength and the fluorescence intensity of fluorescent probes [36,37]. In protic solvents such as ethanol and water, intermolecular hydrogen bonds between the nitrogen atom of the aniline group and the -OH groups of the solvents may occur and stabilize the ground state [38,39]. When the polarity of the solvent increases, the fluorescence quenching is generally attributed to charge/proton transfer or electron transfer [37,38,40]. Therefore, in the next experiments, an ethanol/water mixture (10/90, v/v) was used as food simulant for lipid-rich products such as fish, meat or cheese, according to the EU regulation n° 10/2011. The same parameters as those previously evaluated in ethanol were analyzed in order to determine the impact of the nature of the solvent on the fluorescence property of the material.

### 3.2.2. Influence of the polarity of the solvent on the kinetics of fluorescence quenching of the material after interaction with the analytes

As depicted in Fig. 5 (Table b), the rate of adsorption of the analyte by the material was assessed in an ethanol/water mixture (10/90, v/v) and compared to that obtained in ethanol. For aldehydes, it was
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Influence of the concentration of the analyte on the percentage of fluorescence loss of the material, limit of detection and saturation limit. a. b. Influence of the concentration of aldehydes (a) and ketones (b) on the percentage of fluorescence loss of the material. I₀ represents the initial fluorescence intensity at 337 nm of 10 mg material in suspension in ethanol. I_c represents the fluorescence intensity of the material at 337 nm after 10 min of stirring of the suspension of the material in the carbonyl solution in ethanol at concentration c. Error bars represent the standard deviations obtained from three replicates. Solid lines are guides for the eyes. c. d. Limit of detection and saturation limit of each carbonyl. Error bars represent the standard deviations obtained from three replicates. Statistical comparisons were performed using one-way ANOVA tests. Significantly different values (p < 0.05) are indicated with different number of stars.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Influence of the presence of an interfering compound (n-hexane) on the fluorescence property of the material. a. Kinetics of the fluorescence decrease of the material after interaction with hexanal and an alkane structural analogue, n-hexane, playing the role of interfering compound. Error bars represent the standard deviations obtained from three replicates. b. Influence of the concentration of hexanal and n-hexane on the percentage of fluorescence loss of the material. Error bars represent the standard deviations obtained from three replicates.</figcaption></figure>

observed that the fastest fluorescence quenching occurred in the following order: propanal > hexanal > nonanal. Thus, the shorter the molecule was, the faster the equilibrium state was reached. In ethanol, the opposite behavior was observed. This could be explained by the fact that polar molecules are more soluble in polar medium and are thus more likely to be conveyed up to the material compared to more

hydrophobic molecules. Moreover, concerning ketones, 1-octen-3-one quenched more rapidly the fluorescence of the material compared to 1-penten-3-one. These molecules did not follow the trend of their aldehydes' counterparts. This is probably due to the mixture of molecules derived from 1-penten-3-one which did not permit any comparison between the two ketones.
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Influence of the solvent polarity on the fluorescence of the material (kinetics of the fluorescence decrease, percentage of fluorescence quenching, limit of detection and saturation limit of each carbonyl). **a.** Evolution of the fluorescence spectrum of a 10 mg suspension of material in four dispersing phases of increasing polarity (ethanol; ethanol/water, (50/50, v/v); ethanol/water, (10/90, v/v) and water). **b.** Kinetics of the fluorescence decrease of the material after interaction with carbonyls in ethanol (EtOH) and ethanol/water (10/90, v/v) mixture. **c.** Percentage of fluorescence quenching of 10 mg of material by carbonyls, in ethanol and ethanol/water (10/90, v/v) mixture at the same carbonyl concentration (9.98 × 10⁻³ mol·L⁻¹). **d.** Limit of detection (d) and saturation limit (e) of the different carbonyls in ethanol and ethanol/water (10/90, v/v) mixture. (n.a.: not applicable due to solubility limitations).</figcaption></figure>

### 3.2.3. Influence of the polarity of the solvent on the fluorescence quenching of the material by the analytes

As displayed in Fig. 5 (Table c), the fluorescence of the material was more efficiently quenched by aldehydes with longer aliphatic chains at a given concentration. Nonanal accounted for 86 % of quenching while propanal only quenched 42 % of the fluorescence at the same concentration (9.98 × 10⁻³ mol·L⁻¹). The ranking of the aldehydes responsible for the most important quenching of fluorescence was as follows: nonanal > hexanal > propanal. Except for propanal, the percentage of fluorescence quenching was higher with each analyte in the ethanol/water mixture (10/90, v/v) compared to ethanol. This is probably due to interactions between the material and the more polar solvent, resulting in non-radiative relaxation processes of the excited material.

### 3.2.4. Influence of the polarity of the solvent on the limit of detection and saturation limit of the sensor

The influence of the concentration of the different analytes on the fluorescence of the material was also assessed in the ethanol/water mixture (10/90, v/v) and compared to the results obtained in ethanol. As 1-octen-3-one was not soluble in the ethanol/water mixture, it was no longer considered for this experiment. As shown in Fig. 5 (Table d), the limit of detection of all of the carbonyls tested was higher in the ethanol/water mixture compared to ethanol. The saturation limit was rather difficult to estimate in the ethanol/water mixture due to the limitation brought by the solubility of some carbonyls (especially nonanal) at high concentrations. As depicted in Fig. 5 (Table e), the saturation limit was lower in the ethanol/water mixture. The fluorescence extinction was thus reached at lower concentrations of carbonyls (propanal, hexanal and 1-penten-3-one) in the ethanol/water solvent. Given the facts that the fluorescence intensity of the material decreases after interaction with the analyte and that the initial fluorescence of the material is lower in more polar solvents, the detectable range of carbonyl concentration is

therefore more limited in ethanol/water mixtures. However, even if the range of detection of the sensor in water-rich solvents is reduced, it is still large enough for the monitoring of lipid oxidation among food products. Actually, food products may release from a few µg·g⁻¹ to a few mg·g⁻¹ of each carbonyl during the oxidative spoilage [16,41,42]. Moreover, it can be considered that around 40 carbonyls molecules are commonly released by lipid-rich products, among which the five tested in this study. Considering an average food product in its packaging, it could be estimated that in the worst-case scenario, no more than 0.25 mol·L⁻¹ of carbonyls should be released. This corresponds to the saturation limit of the sensor presented. Saturation limit is thus high enough to monitor the global content of carbonyls released during the lipid oxidation of a food product. This brings an additional proof that the material could also be successfully used for carbonyl monitoring in water-rich products.

### 3.3. Influence of the pH on the fluorescence property of the material

When 10 mg of material was placed in suspension in 2 mL of ethanol, the pH was 7.82 ± 0.02. The impact of the pH of the dispersing phase on the fluorescence property of the material was also studied. Fig. 6 depicts the fluorescence intensity of the material at its maximum emission wavelength (337 nm) before and after interaction with 1-octen-3-one according to the pH of the ethanol/water (10/90, v/v) mixture in which the material was placed in suspension. At pH 1.4, the fluorescence intensity of the material was close to zero. Under acidic conditions, the aniline is protonated (pKa₁ = 4.6) and the anilinium ion is non-fluorescent [43]. This is probably due to the loss of the lone pair of electrons from the nitrogen atom resulting in reduced delocalization of the π-electrons over the benzene ring, which is responsible for the initial fluorescence of aniline. As the fluorescence of the material is extremely weak at this pH, it cannot be further quenched by interaction with 1-
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Influence of the pH of the dispersing phase on the fluorescence property of the material. Evolution of the fluorescence intensity at 337 nm of 5 mg of material before (blue empty bar) and after (red hatched bar) interaction with 1-octen-3-one for 10 min, according to the pH of the ethanol/water (10/90, v/v) dispersing phase. Error bars represent the standard deviations obtained from three replicates. Corresponding theoretical evolution of the structure of the aniline group of the sol-gel sensing material according to the pH. m and n are the molar ratio of functional monomer and cross-linker used for the synthesis of the functionalized sol-gel sensing material, respectively.</figcaption></figure>

octen-3-one. Moreover, protonated amines exhibit reduced nucleophilic reactivity. Thus, 1-octen-3-one did not have any impact on the fluorescence of the material at this pH. Similar behavior was observed at pH 12.8. Aniline was deprotonated (pKa₂ = 9.3) and the material became non-fluorescent. In such a case, the aniline anion is probably not fluorescent because of charge/proton transfer non-radiative relaxation process. Moreover, a dissolution of the silicate-based material started to occur under basic conditions. This phenomenon was already noticed at pH > 9 by Krauskopf [44]. It could be related to the polarization of Si-O bonds in the bulk by the negatively charged Si-O groups on the surface of the material, leading to a weakening of the Si-O bonds promoting the dissolution of the silica-based material [45]. At pH 7.6 and 9.3, the fluorescence intensity of the material was at its maximum. This is in good agreement with the observations previously made by Williams, who highlighted that the maximum fluorescence of anilines was obtained at a pH between 7 and 10 [43]. Therefore, aniline grafted onto a silica structure seemed to respond to pH variations in a similar manner as aniline in solution. Over this pH range, the fluorescence of the material was significantly quenched by 1-octen-3-one. At intermediate pH, the amino group of the aniline is not charged. Molecular aniline is thus both highly fluorescent and highly reactive compared to its ionic forms. According to these results, the optimal conditions for using the material are at a pH between 7 and 9. Such material could thus be used for the monitoring of lipid oxidation for a lot of different food products, as usual pH of common lipid-rich products (milk [46], butter [46], fish [47], meat [48]) is around 6–7.

## 4. Conclusion

To conclude, the performances of a new fluorescent material

intended to be used as a sensor for the monitoring of carbonyls secondary products of lipid oxidation was assessed. The impact of the nature of the carbonyl, including the aliphatic chain length, as well as the solvent polarity and its pH on the fluorescence property of the sol-gel sensing material were investigated. The designed material exhibited high fluorescence that was quenched upon interaction with carbonyl compounds. In ethanol, the kinetics of fluorescence decrease induced by the interaction with the tested carbonyls (propanal, hexanal, nonanal, 1-penten-3-one and 1-octen-3-one) was fast (less than 5 min). The molecular size and the hydrophobicity of the analyte seemed to be the major parameters that affected the adsorption rate, the percentage of fluorescence quenching as well as the limit of detection and saturation limit of the sensor. The presence of interfering compounds, n-hexane and *n*-octane selected as structural analogues of the carbonyls tested, had a limited contribution to the quenching of the fluorescence of the material. This will have to be taken into account in the analysis of complex matrices if such molecules are obviously present in the matrix studied. In more polar solvents, the fluorescence intensity of the material was lower because of charge/proton transfer or electron transfer mechanisms. However, the kinetics of fluorescence decrease remained fast (less than 5 min), and the fluorescence quenching by carbonyls was significant enough to detect carbonyls over a wide range of concentrations (from 0.6 mmol·L⁻¹ to 0.27 mol·L⁻¹). The optimal pH range for the use of the sensor was 7–9, corresponding to molecular form of the aniline, conferring to the material both high fluorescence and high reactivity towards carbonyls. The sensor was thus proven to be efficient in different food simulants, at pH corresponding to most of the lipid-rich food products, and with a range of detection allowing the lipid oxidation to be monitored all along the shelf-life of the food product. Such a sensor could therefore be used to assess lipid oxidation levels in liquid
matrices more rapidly, safely, and easily than traditional methods. Future work will focus on the application of the newly developed sensor on real food systems, starting with oils and following with more complex matrices.

### CRediT authorship contribution statement

**Laura Carballido:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Virginia Guerlet:** Validation, Investigation, Formal analysis, Data curation. **Elias Bou-Maroun:** Writing – review & editing, Visualization, Validation, Supervision, Methodology, Funding acquisition, Formal analysis, Conceptualization. **Thomas Karbowiak:** Writing – review & editing, Visualization, Supervision, Resources, Funding acquisition, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Data availability

Data will be made available on request.

### Acknoledgements

This work was financially supported by the French "Investissements d'Avenir" program, project ISITE-BFC (contract ANR-15-IDEX-0003) and related the patent n WO2023110783. The research leading to these results has also received funding under grant CAPTIV from Carnot Qualiment© (DOI: 10.17180/h5gd-gk88) supported by Agence Nationale de la Recherche. The authors acknowledge the DIMACELL platform (Institut Agro Dijon) for the access to the fluorescence spectroscopy equipment.

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