Optimized erucic acid-based extract as a natural probe for viscosity detection of liquid safety

2.1 Materials and apparatus

The chemical reagents in this study were directly obtained from Macklin Biotechnol-ogy (Shanghai) Co., Ltd, and Shanghai Aladdin Bio-Chem Technology Co., Ltd. All the reagents and solvents were used without any further purification. Milli-Q water purifica-tion system (Millipore, Bedford, MA, USA) was used to prepared the deionized (DI) water. Nuclear magnetic resonance (NMR) spectra were obtained with Bruker AVANCE III HD 600 NMR Spectrometer, with TMS internal standard in DMSO‑d₆. High resolution mass spectra (HR-MS) were performed through Aglient 7250 & JEOL-JMS-T100LP AccuTOF mass spectrometer. Fluorescence spectra were recorded by a Hitachi F-7000 fluorescence spectrophotometer. Absorption spectra were detected on a Hitachi U-3010 UV–Vis spectrophotometer. The viscosity determination test was performed on a rotating viscometer (DV2T, Brookfield, AMETEK Corp., USA). The reagents and instruments were described in the Supplementary Material.

2.2 Preparation of plant extract erucic acid (EAd)

Natural rosemary was freeze-dried as the powder, then soaked into 80 % ethanol with continuous shaking at 80 °C for 3 h. The solution was led to filtration, ethanol extract was concentrated under vacuum through the rotary evaporator. The obtained product was dissolved in distilled water and partitioned with n-hexane. Controllable fractions of n-hexane and water mixture were utilized, and obtained organic solution was evaporated under vacuum. Afterwards, the obtained extract was purified by flash column chromatography over silica gel to give the erucic acid (EAd) as a pale yellow solid, yield 1.1 %. ¹H NMR (600 MHz, DMSO) δ 12.13 (s, 1H), 8.90 (s, 1H), 7.51 (d, J=15.9 Hz, 1H), 7.00 (s, 2H), 6.43 (d, J=15.9 Hz, 1H), 3.81 (s, 6H). ¹³C NMR (101 MHz, DMSO‑d₆) δ 168.42, 148.53, 146.52, 138.57, 125.09, 116.55, 106.59, 56.57. HRMS (ESI): m/z calculated for 224.21200, found: 225.21204 [M+H]⁺.

2.3 Measurements of optical properties

The molecular probe EAd was dissolved in DMSO to prepare the stock solution with the concentration of 1 mM, and stored under the temperature of 5 °C before the test. EAd solutions (10 μM) was added into different volume fractions of distilled water and glycerol mixtures (from 0 % to 99 %), and each sample were recorded by the fluorescence spectra and UV–Vis. To prepare different polarities of liquid system, the common solvents including the EtOAc, methanol, THF, toluene, etc. were selected to test the emission properties. In the specificity test, the solutions with various potential relevant analytes (including NaCl, K₂CO₃, VC, sodium benzoate (SB), etc., 50 μM) were prepared with distilled water, and EAd solutions were added as well. The fluorescence emission spectra were obtained by the fluorescence spectrometer. In the temperature effect experiment, EAd was diluted with the glycerol under different temperatures, including the normal body temperature (37 °C), fresh-maintenance temperature (5 °C) and ambient temperature (25 °C). In all measurements, the mixed solutions were transferred to the quartz cell. The excitation wavelength was set as 320 nm.

2.4 Viscosity tracking in the spoilage process

The raspberry fruit juice and lemon liquids were brought from the local supermarket, and flesh floats and sediments were removed. Before the storage process, the bubbles were removed as well, and shaked thoroughly with the transparent glass bottle. Then, both fresh liquids were stored at different temperatures (25 °C, 5 °C & 37 °C) when exposed to the air for 7 days. During the storage process, the fluorescence emission spectra were recorded within this time interval (at day 0, day 2, day 4, and day 7) with the addition of molecular probe EAd dilution (10 μM). The relationship between fluorescence signal intensity and viscosity value has been established: (F_n-F₀)/F₀∼(η_n-η₀)/η₀, F_n & F₀ fluorescence intensity at day n and day 0, η_n & η₀, viscosity value at day n and day 0.

2.5 Theoretical calculations

The calculations were performed using the Gaussian 09 program, ground state structure of the rotor was optimized using time-dependent density functional theory (TD-DFT) by using the B3LYP/6-31G(d) level of theory. The energy gap and oscillator strength fem were calculated as well.

3.1 Molecular probe selected and extracted strategy

Several kinds of natural products can be used as molecular tools for physical micro-environmental viscosity sensing, the ingenious selection of a powerful optical probe is highly desirable. The natural extract EAd was found to bear phenolic hydroxyl and carboxyl groups together with the conjugate single and double bond, which exhibited a flexible and rotatable chemical structure. Through the linkage with D and A, this kind of natural probe might display different emission fluorescence and sensing properties toward viscosity via the potential rotation of conjugated bond. In the low viscosity media, weaker fluorescence emitted, while in the higher viscosity liquid, stronger signal came out, a turn-on mode can be found. We hypothesized that the signal change phenomenon was attributed to the restriction of rotatable part, in normal commercial liquids, the rotation is free, rapid consumption of energy may lead to weaker fluorescence, while in spoiled liquids, the rotation is restricted, and the energy consumption by the non-radiative pathway will be reduced, and the transition of TICT and ICT was activated, detailed chemical structure and sensing mechanism was shown in Scheme 1. Thus, the capability of viscosity sensing was existed objectively. In detail, the molecular probe EAd was extracted from the rosemary and has been purified through the silica gel, which was utilized directly. The chemical structure and detailed relative molecular mass of probe EAd were defined by ¹H NMR, ¹³C NMR and high-resolution mass spectroscopy (HR-MS), as presented in Fig. S1-Fig. S3 (in the Supplementary Material).

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Scheme 1

Chemical structure of natural probe EAd and sensing mechanism.

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3.2 Responsive properties toward viscosity

The above selected strategy intrigued us to investigate the optical responsive proper-ties toward viscosity. Initially, the fluorescence and absorption spectroscopic properties of natural probe EAd were investigated. As shown in Fig. 1a and 1b, a remarkable fluorescence signal can be observed in the high viscous glycerol, 52-fold enhancement was clearly found. Moreover, the physical properties of quantum yields and fluorescence lifetime in low viscosity water and high viscous glycerol have been collected as well, as shown in Table S2. There is nearly no fluorescence emission intensity in the water, and this remarkable difference in the intensity suggests that natural probe EAd could serve as a molecular tool for viscosity detection. Comparatively, natural probe EAd exhibited an ab-sorption peak at 329.3 nm in the high-viscosity glycerol, which was slightly red-shifted from the peak 324.9 nm in the lower viscosity pure water. The parallel stacking of molecular probe in the glycerol and the conjugation may be enlarged (Ge et al., 2023; Zhang et al., 2024). Furthermore, the viscosity-responsive properties of natural probe EAd was explored in different volume fractions of glycerol and water mixtures, as shown in Fig. 1c. With the increment of glycerol, the emission intensity increased gradually, which may be attributed to the inhibited rotation of conjugated bond in the viscous environment. Moreover, a good linear relationship (R² = 0.98) of the maximum emission intensity against viscosity from 1.0 cP to 956.0 cP was observed by fitting the Förster–Hoffmann equation, as shown in Fig. 1d (Wang et al., 2023). The viscosity-sensitive coefficient (x) was calculated as 0.56, and the detection limit was determined to be 1.253 cP by using the commonly equation LOD=3 σ/S, where σ means the standard deviation of a blank and S represents the slope of calibration line, as shown in Fig. S4 (Ma et al., 2024). These detailed results further confirmed natural probe EAd can potentially serve as an excellent viscosity-responsive molecular tool in a relatively large viscosity range. Besides, the viscosity sensing capability under different temperatures were investigated, since temperature is one of the key factors that can affect the viscosity. It can be found that the fluorescent intensity increased when the glycerol was stored under lower temperature, while the signal became weaker when under a higher temperature, as shown in Fig. S5. The test results further illustrate that natural probe EAd can potentially work as a sensitive molecular tool for viscosity tracking in solutions. In order to confirm the applications in commercial liquids, we tested natural probe EAd in ten kinds of liquids, including water, pomelo juice, pear juice, raspberry fruit juice, milk, lemon juice, mango juice, watermelon juice, edible oil, and glycerol, as shown in Fig. S6. Detailed fluorescent intensities in those liquids were collected in Table S3. In addition, the viscosities of these commercial liquids were measured by the viscometer as well, and the calculated results in Table S4 were inconsistent with those by the fluorescent method. We can clearly find that various fluorescence intensities in these commercial liquids, suggesting the natural probe EAd showed enormous potential to be utilized in the commercial complex samples.

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Fig. 1

(a) Fluorescence spectra of the natural probe erucic acid (EAd) (10 μM) in water and glycerol. (b) Absorption spectra of the natural probe erucic acid (EAd) (10 μM) in water and glycerol. (c) Fluorescence emission spectra of the natural probe erucic acid (EAd) (10 μM) in the water/glycerol mixture with different glycerol volume fraction of 0 %∼99 %). (d) The linear relationship between log I_max and log η.

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The thickeners can enhance the consistency, homogeneity and stability in commercial liquids, and have been one of the main components as food additives (Pirsa and Hafezi, 2023). And therefore, we further explored the sensing capability of natural probe EAd with the existence of three kinds of food thickeners, such as SCC, Pec, and XG. As shown in Fig. 2a, the fluorescence spectra were first recorded with the addition of various amounts (1.0 g/kg-5.0 g/kg) of above-mentioned thickeners. Fluorescent intensities were gradually increased with adding amounts of the food thickeners, viscous media was formed in a certain way. In Fig. 2b, it is worth to mention that a positive relationship was existed between the mass concentrations of food thickeners and fluorescence intensities. And we can find that its fluorescence in the xanthan gum (XG) reached the highest, indicating that XG exhibited the best thickening efficiency, while the fluorescence in sodium carboxymethyl cellulose (SCC) occupied the lowest, which means the thickening efficiency of SCC is lower. Therefore, we can conclude that the natural probe EAd can be appropriately acted as the intelligent fluorescent probe toward viscosity identification.

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Fig. 2

(a) Emission spectra of the molecular probe EAd in the SCC, Pec, and XG solutions with the addition amount from 1 g/kg to 5 g/kg. (b) Histogram of signal intensity of EAd at different viscosity of media, and fitting line with the mass amounts of SCC, Pec, and XG. The concentration of the molecular sensor EAd = 10 μM, λ_ex = 320 nm.

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3.3 Photophysical properties investigation

Next, the photophysical properties of natural probe EAd in relation to the stability in various liquids with different polarities were investigated. Herein, the fluorescence emission and absorption spectra of EAd in eight kinds of common solvents with different dielectric constants (ε) were investigated. A remarkable fluorescence signal peak at 420 nm was found in the emission spectra of high-viscosity glycerol, as shown in Fig. 3a. And the intensities of emission spectra in other low viscous solvents were found weaker. The absorption spectra in these common solvents were distributed around 320 nm. Negligible peak wavelength can be observed, as shown in Fig. 3b. Corresponding fluorescent im-ages were exhibited in Fig. 3c, bright emission signal can be observed only in the glycerol, while weak signal was released in other liquid samples. Significant differences can be visualized from the naked-eye. High molar extinction coefficient in the water can make it possible to provide a low fluorescence background in liquid imaging. Detailed photophysical properties of the sensor EAd in these solvents were collected in Table S5. These test results indicated that the fluorescence signal cannot be affected by the solvent polarity. And the stability in various solvents was extremely well, which can be helpful to avoid the complex polar influence.

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Fig. 3

(a) Fluorescence emissive spectra of the molecular probe EAd in various common solvents. (b) Absorption spectra of the molecular probe EAd in various common solvents with different polarities. (c) Fluorescent images in various solvents. (d) Emission spectra of EAd (10 μM) in various solutions with the potential analytes in liquid food. (e) Selectivity of the probe EAd (10 μM) toward various liquids-related analytes, including blank, Na⁺, K⁺, Ca²⁺, SO₄^2-, Cl^-, NO₃^–, CO₃^2–, gallic acid, acesulfame, sorbic acid, vitamin C, erythorbic acid, sodium benzoate (SB), beet molasses (BM), trisodium citrate dehydrate (TCD), and glycerol. Concentration of the analyte is 50 μM.

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Afterward, the selectivity and anti-interference of natural probe EAd to various potential analytes in the liquid system were investigated, since the micro-environment in the liquid system is complex. With the inclusion of ions (Na⁺, K⁺, Ca²⁺, SO₄^2-, Cl^-, NO₃^–, CO₃^2–), common liquid contents (gallic acid, acesulfame, sorbic acid, erythorbic acid, sodium benzoate, beet molasses, trisodium citrate dehydrate) and vitamin C, the FI response intensity of EAd was extremely lower. As shown in Fig. 3d and 3e, negligible changes can be found in the fluorescence intensity histogram with these additives. Only dramatically enhanced fluorescence signal was triggered by the glycerol, which showed that the fluorescence signal cannot be influenced by the various food additives, and FI response of EAd to viscosity (i.e., glycerol) was higher than for other species. Moreover, the photostability in different pH has been evaluated, as shown in Fig. S7, six kinds of buffer solutions in the pH range of 3.0–9.0 were prepared, with the final concentration of 10 μM. The emission intensities were found to be even lower, a negligible influence on the signal emission intensity during the test common pH range. And the pH stability under high viscous glycerol has been tested as well, In addition, the photostability of natural probe EAd among various commercial liquids were tested as well. As shown in Fig. S9, higher stability of fluorescence intensity was maintained under 60 min continuous light irradiation, and no obvious attenuation phenomenon was occurred among these commercial liquids. As presented in Fig. S10, it can be found that the maximum stokes shift of EAd in water varied from 324.9 nm to 434.3 nm, and the stokes shift in glycerol distributed from 329.3 nm to 412.6 nm, respectively. It showed a large stokes shift of 109.4 nm and 83.3 nm, respectively, which may avoid the overlap of absorption the auto-fluorescence and enhance the signal-to-noise ratio (Wang et al., 2024). The results are better than most of the reported studies as listed in Table S1. These data revealed that EAd owns high photostability, excellent chemical stability, and large stokes shift, which suggesting its feasibility of applications in complex liquid system.

3.4 Theoretical calculation

The response mechanism of natural probe EAd to viscosity was further identified, a theoretical calculation based on DFT/B3LYP/6-31G was performed using Gaussian 09 software (Ndaleh et al., 2024). As shown in Fig. S11, the HOMO and LUMO energy levels for ground and excited states of natural probe EAd were measured. The HOMOs were found to be mainly located at phenolic hydroxyl group, while the LUMOs were basically located on at carboxyl group. The energy gaps of natural probe EAd at different rotation states were calculated to be ΔE=6.2159 eV at 0° and ΔE=6.8876 eV, respectively. The HO-MO-LUMO gap in excited state was smaller than that in ground state, which may lead to strong charge transfer and the occurrence of an ICT process. In the high viscous micro-environment, the molecule EAd rotation may be restricted and fluorescence intensity was enhanced due to the transferring between different rotation angels. Moreover, the oscillator strength fem values were calculated to be 0.6538 at 0° and 0.0033 at 90°, the dihedral angels of EAd under excitation state and ground state were quite different, since the conjugate structure of EAd in the excited state was well planarized, and its planarity was destroyed with the rotational motion back to the ground state. These results indicated that intramolecular rotation of EAd was exhibited when the micro-environmental viscosity became lower. Furthermore, the solvatochromism of the natural probe EAd was tested in six kinds of representative solvents, as shown in Fig. 4. Typical red shifting phenomenon in both absorption and emission spectra can be observed, which may be ascribed to the stabilization of ICT process occurring between the phenolic hydroxyl group and carboxyl group (Zheng et al., 2024). These tests confirmed that the intra-molecular rotation of conjugated molecule EAd can form a typical twisted excited state.

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Fig. 4

(a) Normalized absorbance of molecular probe EAd in six presentative solvents. (b) Normalized emission spectra of molecular probe EAd in six presentative solvents.

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3.5 Deterioration process monitoring

To investigate its performance in liquid systems, the natural probe EAd was employed for the detection of viscosity fluctuation in the representative liquid system. Thus, two kinds of commercial liquids named raspberry fruit juice and lemon juice were selected to test its tracking capability during the spoiled process. In detail, these liquids were stored under ambient temperature and lower-maintenance temperature for 7 days, respectively. Corresponding digital images were displayed in Fig. 5a.When the liquids were stored under ambient temperature, at the beginning of two days, both liquids were trans-parent and clear appearances, and the color has not dimmed yet. After day 4, the color of raspberry fruit juice has been faded, bleached phenomenon can be observed, especially at day 7. And several kinds of floating objects occurred in the lemon juice and turbid phenomenon became seriously. On the contrary, when these two kinds of liquids were stored under lower-maintenance temperature, a normal appearance and transparent phenomena can be observed even though the storage time was extended to 4 days, as shown in Fig. 5b. After day 4, the apparent color of raspberry fruit juice was still retented, and only muddy phenomena can be found in the lemon juice. During the process of monitoring the deterioration of raspberry fruit and lemon juice, with the increase time of extension storage under ambient temperature and lower temperature, the emission signals have been tracked along the 7 days with spectrometer. And the whole deterioration process was measured at day 0, day 2, day 4 and day 7. Before the test, all the samples were pretreated with the natural probe EAd with the concentration of 10 μM, and then led to the fluorescence investigation. The results were shown in Fig. 5c and Fig. 5d, it can be found that the FI signal enhanced obviously with the storage time extended, 17.3 % and 20.2 % increment were found after 7 days under ambient temperature in the raspberry fruit and lemon juice, respectively. However, only 9.1 % and 10.6 % enhancements can be found when the raspberry fruit and lemon juice were stored under lower-maintenance temperature. Moreover, both fresh liquids were stored under high temperature (37 °C) as well, as shown in Fig. S12. It can be found that more severe deterioration phenomenon and higher emission enhancements occurred, 21.3 % and 26.9 % enhancements were found in the raspberry fruit and lemon juice, which indicated that higher temperature can accelerate the deterioration process. These data confirmed that lower temperature can slow down the deterioration speed, and extend the storage time. More importantly, the abovementioned results indicated that the molecular probe EAd can monitor the micro-environmental viscosity fluctuation, and can be utilized as an out-standing luminescent tool to achieve deterioration extent sensing.

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Fig. 5

(a) Digital images of the raspberry fruit juice and lemon juice stored under ambient temperature within 7 days. (b) Digital images of the raspberry fruit juice and lemon juice stored under low-er-maintenance temperature within 7 days. (c) The emission intensity enhancement of raspberry fruit juice and lemon juice during the 7 days under ambient temperature. (d) The emission intensity enhancement of raspberry fruit juice and lemon juice during the 7 days under lower-maintenance temperature. The concentration of EAd = 10 μM, λ_ex = 320 nm.

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In order to consolidate this conclusion, we studied the change pattern of viscosity values via the viscometer as well. As seen from Fig. 6a and Fig. 6b, the viscosities of raspberry fruit and lemon juice increase sharply after day 2 when stored at ambient temperature. 36.4 % and 44.8 % enhancements were tested in the raspberry fruit and lemon juice, respectively. By contrast, the viscosities of raspberry fruit and lemon juice enhanced only 17.7 % and 26.5 % when stored at the lower-maintenance temperature. The results were consistent with above fluorescence intensity results. Notably, a fitting linear relationship was established between the viscosity increment percentage (η_n-η₀)/η₀ × 100 % and the fluorescence intensity increment percentage (F_n-F₀)/F₀ × 100 %, as displayed in Fig. 6c. With the combination of fluorescence spectra and traditional viscosity determination results, it can be concluded that the micro-environmental viscosity became higher during the deterioration process, and normal ones can be distinguished from the spoiled ones through the fluorescent technique.

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Fig. 6

(a) Viscosity values of raspberry fruit juice when stored under different temperatures. (b) Viscosity values of lemon juice when stored under different temperatures. (c) The fitting line among the fluorescence increment percentage and viscosity enhanced degree.

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