Investigation of the use of carbon dots from Euphorbia amygdaloides for the detection of aflatoxin B1

2.1. Chemicals

Hydrochloric acid (HCl), sodium hydroxide (NaOH), urea (CH₄N₂O) (Merck), methanol (CH₃OH), and ammonium citrate (C₆H₁₇N₃O₇) (Merck), from Sigma‒Aldrich, AFB1 mg mL^-1 were purchased.

2.2. Carbon quantum dots synthesis

The pyrolysis technique was used to synthesize CDs from EA. The pyrolysis technique offers several advantages, including non-toxicity, simplicity, reproducibility, low cost, and easy accessibility. Furthermore, it allows for precise control of reaction parameters such as temperature and pH, resulting in the production of high-quality CDs. Importantly, this non-toxic approach increases the biocompatibility of the resulting EA-CDs, making them suitable for the sensitive and safe detection of AFB1.

The EA plant was collected from the garden of the Technical Sciences Vocational School, Erzurum Atatürk University, Turkey, and identified by the Department of Biology, Faculty of Science. EA plant samples were first washed, dried, and then crushed with liquid nitrogen in a mortar. CDs were synthesized using the Polat and Nadaroglu method [6]. Lastly, EA-CDs were kept at +4°C until the date of operation.

The quantum yield of EA-CDs was found as %QY=%54 (reference quinine sulfate) [9]. The experimental process has been summarized in Figure 1.

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Figure 1.

Experimental setup design for the development of fluorescence resonance energy transfer mechanism (FRET) based sensor for the detection of AFB1 using EA-CDs.

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2.3. Investigation of the interaction between EA-CDs with AFB1

To evaluate the changes in fluorescence intensity during the interactions of AFB1 with EA-CDs, fluorescence measurements of the EA-CDs solutions (20 ppb) were taken at increasing AFB1 concentrations in the range of 2.5 × 10^-7 - 2 × 10^-6 ppm 0.25-2 ppb. Firstly, they were incubated for 40-60 min (25°C), and then the fluorescence intensities were measured depending on the excitation wavelength. In addition, different parameters such as temperature, pH, and incubation time were also tested to determine the AFB1 binding optimization with EA-CDs.

2.4. Limits of detection and quantification of AFB1 in food samples

Using EA-CDs synthesized via pyrolysis, AFB1 was identified in some food samples (peanut, corn, and milk). Peanut from the food samples used in the experiment was purchased from local markets in Osmaniye, Turkey. Milk and corn samples were obtained from local markets in Erzurum, Turkey. The samples were briefly prepared as follows: 8 g of peanut or corn samples were taken into a mortar and crushed with liquid nitrogen. It was prepared by adding a methanol/water mixture (40 mL; 8:2, (v/v) ratio) and was stirred at 450 rpm using a magnetic stirrer at room temperature for 1 h. Firstly, it was centrifuged at 5000 × g for 30 min. Then, it was taken and stored at +4^oC until use. The milk fat was first separated by centrifugation (9000 × g, 25 min), and then the samples were prepared by adding 5 mL of a methanol/water mixture at the same ratio to 1 mL of milk. Then, the known concentration of AFB1 and EA-CDs fluorescence intensity was measured.

3.1. Characterization of the synthesized EA-CDs

Urea was used as a reducing agent and ligand in the preparation of our EA-CDs. EA-CDs were obtained equipped with –NH groups and showed high fluorescence properties with superior stability. Moreover, it has the advantage of being synthesized without containing toxic materials (Figure 2).

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Figure 2.

Typical TEM image of EA-CDs.

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The morphological analysis of EA-CDs from transmission electron microscopy (TEM) images has been shown in Figure 2(a). It was determined that EA-CDs have a uniform, spherical morphology. The size distribution was determined to be in the range of 2.23 ± 0.14 nm to 10.12 ± 1.04 nm (Figures 2a and b). The particle dimensions sequenced from almost 1.20 to 3.12 nm, and the mean dimension was 2.05 nm. High resolution transmission electron microscopy (HRTEM) images showed an interlayer lattice spacing of 0.22 nm [10].

The 2-10 nm particle size range is an important parameter that directly affects the optical properties and detection performance of EA-CDs.

As the size of the CDs decreases, the quantum confinement effect becomes more pronounced, leading to an increase in the band gap energy and, consequently, the emission of higher-energy (blue-shifted) photons. This increases the photoluminescence intensity of EA-CDs and improves the signal-to-noise ratio, enabling the highly sensitive detection of AFB1 at low concentrations. Furthermore, the small particle size provides a large surface-to-volume ratio, enabling the presence of numerous surface functional groups that facilitate stronger and more selective interactions with AFB1 molecules, thus increasing both sensitivity and selectivity in the detection process. A feature of the photoluminescence (PL) for CQDs is the region λ_ex that varies with the emission wavelength and intensity. The EA-CDs obtained in our study exhibit blue-green emission in the range of 430–460 nm, consistent with the emission spectra of most plant-derived CDs.

3.2. FT-IR analysis

Functional groups of EA-CDs were shown using the FTIR spectrum (Figure 3a). While the large peak in the 3400–3100 cm⁻¹ range was associated with hydroxyl groups (–OH), the vibrations of -NH₂ groups were also associated with the vibrations in this range. The vibration was associated with C=O at 1595 cm⁻¹, and the peak at 1498 cm⁻¹ was associated with C=C vibrations. It shows the presence of carbon structures in aromatic ring structures or sp² hybridization in graphite structures. While aliphatic -C-H in alkane structures shows stretching vibrations at 2887 and 2523 cm⁻¹, peaks at 1130 and 1200 cm⁻¹ indicate the presence of ether or alcohol groups (C-O-C, C-OH, or C-O) [11]. All these peaks are clear indications that the surfaces of EA-CDs are functionalized. This is a clear indication that the structure has gained hydrophilic character and can exhibit a stable structure in aqueous solution.

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Figure 3.

(a) FTIR analysis, (b) XRD version (c) XPS spectra , and (d) examination XPS spectrum of EA-CDs

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The FTIR spectrum of EA-CDs revealed the presence of –OH, –NH₂, C=O, C=C, C–O–C, C–O, C–N, and C–H functional groups. These surface groups enhance the interaction capacity of EA-CDs with aflatoxin molecules. Specifically, carbonyl and ether groups can participate in electrostatic and π–π interactions, while hydroxyl and amine groups can form hydrogen bonds with aflatoxin. Such specific interactions contribute significantly to the sensitivity and selectivity of the EA-CDs, facilitating efficient capture and detection of aflatoxins.

3.3. XRD analysis

Various peaks of EA-CDs were shown in the X-ray diffraction (XRD) patterns in Figure 3(b), showing a combination of crystalline and amorphous phases. It was shown with a broad peak of 26° corresponding to the graphite (002) planes at a 0.24 nm interval, which belongs to CDs and shows graphite properties. This result shows a layered graphite structure. The results, which are consistent with previous synthesis findings, confirm the formation of graphite-CDs [12]. The obtained EA-CDs with this layered lattice structure will provide the properties that will enable the detection of AFB1, as they provide electrical conductivity and structural stability.

3.4. XPS analysis

The X-ray photoelectron spectroscopy (XPS) survey spectrum shows the presence of three prominent peaks corresponding to the binding energies of C 1s at 286.00 eV, N 1s at 400.00 eV, and O 1s at 531.00 eV (Figure 3c).

The presence of carbon, nitrogen, and oxygen in the EA-CDs structure was confirmed by these peaks, as well as indicating the presence of functionalization groups in EA-CDs. The C 1s peak at 286.00 eV indicates sp² and sp³ hybridizations in EA-CDs. In addition, the EA-CDs structure has a complex network and shows strong luminescence. Thus, it plays a great role in the sensitive and selective detection of AFB1 using EA-CDs. These structures are of great importance in ensuring the strong photoluminescence properties of the synthesized EA-CDs and in sensing aflatoxins. All these improvements made EA-CDs more suitable for the sensitive and selective detection of aflatoxins. [13].

The binding energy at 400.00 eV indicates the N 1s peak, confirming the presence of nitrogen atoms in the functional structures. Nitrogen doping into the EA-CDs structure improved their electronic properties and additionally increased their photoluminescence.

The O 1s peak in carboxyl, carbonyl, and hydroxyl groups is seen at 531.00 eV. Thanks to the functional groups of EA-CDs, the structure is hydrophilic and stable, and they also play an important role in the detection of AFB1. This photoluminescence property of EA-CDs has been shown to be usable for developing precise and selective sensors for the detection of AFB1 [14]. It was also determined by XPS elemental analysis that the structure of EA-CDs has a composition of 61.99% C, 33.71% N, and 4.298% O [14]. It was also determined that the FTIR and XPS analysis results were compatible with each other (Figure 3d).

XRD and XPS were used as two powerful and complementary techniques to verify the crystallinity and chemical composition of EA-CDs. XRD confirmed the presence of graphite-like crystalline regions in carbon-based materials, providing information on the crystal structure and average grain size, while XPS provided information on the oxidation states of surface atoms and chemical bonding environments. Together, these analyses confirmed both the structural order and surface functionality of EA-CDs, providing direct information on their optical properties and surface reactivity.

3.5. Fluorescence and absorbance analysis results

The fluorescence emission spectra of EA-CDs were measured at excitation wavelengths of 300–500 nm. It was observed that EA-CDs exhibited the highest fluorescence intensity at 451.94 nm when the excitation wavelength was 370 nm (Figure 4a).

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Figure 4.

(a) Excitation-dependent FL emission spectra of EA-CDs (b) Excitation-dependent PL emission spectra (c) normalized spectra of diluted EA-CDs, (d and e) UV-Vis absorbance spectra of EA-CDs ranging from 300 nm to 500 nm.

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EA-CDs synthesized based on the pyrolysis method were determined to be intensely blue, and the CDs were light yellow in daylight.The fluorescence intensity was measured at EA-CDs concentrations diluted from 1/15 to 1/60, and the effectiveness of different concentrations on PL was determined. The strongest fluorescence emission intensity was observed when it was diluted at a 1/55 rate (Figure 4b).

In addition, the broad emission peak seen in Figure 4(c) indicates that EA-CDs contain various emitting derivatives. The obtained results have been normalized and given in Figure 4(c). The PL intensity of EA-CDs decreased gradually, and the peak maximum shifted towards the red. These findings are consistent with the literature [15,16].

The analysis of the optical properties of EA-CDs was investigated by measuring absorbance and PL emission spectra. UV-Vis absorption spectra of EA-CDs have been shown in Figures 4(d and e). The peaks around 338 nm belong to the C=C and C=O functional groups, belonging to the π-π* and n-π* transitions [17]. The absorbance graphs obtained from dilutions between 1/55 and 1/60 have been given in Figure 4(e) [18].

The well-defined absorption properties of EA-CDs are an indication of the high quality and purity of our plant-based CDs, and they are suitable for sensors and other applications to be developed based on optical properties.

The effects of pH, temperature, and storage time were investigated to evaluate the absorbance stability of EA-CDs. When the EA-CDs suspension was checked after being stored at +4^oC for 6 months, it was found that there was no turbidity or sedimentation, and it remained stable. When pH is changed, the charge distribution and electron density of the surface functional groups of CDs change. All changes affect the energy levels of the CDs and, consequently, their PL properties.

When the relationship between pH change and fluorescence intensity was investigated, the maximum increase in fluorescence intensity occurred at pH 10 and pH 11, and a blue shift was observed (Figure 5a). The blue shift in the PL indicates the improved quantum yield of EA-CDs in alkaline conditions. The blue shift is owed to the deprotonation of functional groups related to EA-CDs’ surface, reducing electron-donating effects and indicating a denser electronic structure for higher energy transitions. The absorbance change of EA-CDs at different pHs was determined (Figure 5b). It was determined that the measured absorbance change increased when the pH of the environment was adjusted by increasing one unit between pH 2-12. The increase in absorbance was observed to increase the absorbance of EA-CDs at basic pHs. The results obtained indicated that the pH change was sensitive to EA-CDs. The pH change in EA-CDs can cause electronic transitions of π-π* and n-π* in graphite nanodomains by depleting or renewing their valence bands. In this case, the valence bands are depleted or renewed. All these properties can be useful in the design of biological imaging agents and may allow the optical response of EA-CDs to act as a sensor that directly reflects environmental pH changes. Previous studies have suggested that CDs may exhibit pH-sensitive behavior, which is consistent with our observations.

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Figure 5.

(a) At different pHs, PL spectra (pH 2-12) and (b) UV-Vis spectra at different temperature (15-40°C). (c) PL spectra of EA-CDs and (d) UV-Vis spectra (λexc = 370 nm), (e) at different times (15 min-24 h) PL spectra of EA-CDs and (f) UV-Vis spectra.

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Previous studies show that the fluorescence properties of CDs can change significantly with pH changes. Ehtesabi et al. [19] reported that the fluorescence intensity of CDs varies depending on the protonation and deprotonation states of the carboxyl and hydroxyl groups on the surface. Similarly, Guo et al. [20] reported that nitrogen-doped CDs undergo increased fluorescence emission and blue shift due to the deprotonation of the amine groups on the surface under basic conditions. Qin et al. [21] suggested that CDs sensitive to pH changes can be used in biosensor applications. These findings support that the pH-dependent optical properties observed in EA-CDs are a general CD property driven by surface chemistry and environmental interactions. From the findings obtained in the analyses performed on EA-CDs, pH-dependent optical properties can be considered as a general feature of CDs driven by surface chemistry and environmental factors.

The effect of temperature on the fluorescence and absorbance stability of EA-CDs was investigated. The fluorescence and absorption spectra obtained for EA-CDs in the temperature range of 15-40°C have been given in Figures 5(c and d).

In Figure 5(c), the fluorescence intensities of EA-CDs peaked at 15°C. It shows that this temperature provides optimum conditions for electronic transitions of CDs and results in the highest quantum efficiency. This peak in fluorescence intensity at 15°C can be attributed to the balance between radiative and nonradiative recombination processes within CDs. At this temperature, nonradiative processes such as phonon interactions are minimized, resulting in maximum fluorescence efficiency. This behavior is generally consistent with the understanding that temperature reduces nonradiative decay pathways, allowing higher fluorescence outputs. As the temperature increases above 15°C, a gradual decrease in fluorescence intensity is observed. This decrease is likely due to increased thermal energy, which increases nonradiative recombination times, such as phonon interactions and collisional quenching. These processes compete with nonradiative recombination, resulting in a decrease in fluorescence intensity at higher temperatures. The decrease in fluorescence with increasing temperature is a well-documented phenomenon in quantum dots and other fluorescent nanomaterials. Yu et al. observed a significant decrease in the fluorescence intensity of quantum dots with temperature and reported that this was due to the non-radiative relaxation processes that increased at high temperatures [22].

Similarly, Luo et al. (2020) reported that the energy transfer mechanisms changed with increasing temperature, which caused fluorescence quenching. These findings provide an important basis for understanding the effect of temperature changes on the optical properties of quantum dots [23].

Figure 5(d) confirms that CDs are stable with temperature changes, as the absorbance does not change with temperature. The sp²-hybridized carbon atoms in the CDs structure do not undergo structural degradation at high temperatures and maintain their stability. Therefore, the fabricated EA-CDs have good absorbance stability under various conditions, making them ideal colorimetric probes with significant application potential.

To determine the stability of EA-CDs as a sensor for AFB1 detection, PL and absorbance measurements were performed at different incubation times between 30 min and 24 h, keeping the 100 ppb concentration constant, see Figures 5(e and f). The findings indicated that the PL intensity reached equilibrium within 60 min and remained stable for 24 h. No change was observed in absorbance values at all times. All measurements were performed after 60 min. The results show that the PL intensity of EA-CDs is stable over time, demonstrating its suitability for use as a sensor and consistent with literature data [24].

3.6. Selectivity of EA-CDs in the detection of AFB1

AFB1 is a toxic compound with strong carcinogenic effects. In this study, EA-CDs were synthesized using a low-energy, environmentally friendly, non-toxic, fast pyrolysis technique. The synthesized EA-CDs showed a bright blue color.

To analyze the binding ability of the synthesized EA-CDs to AFB1, we first investigated the interaction mechanism of AFB1 with the EA-CDs system. In the measurements, the fluorescence intensities of EA-CDs were measured, and changes were monitored by increasing the AFB1 concentration together with EA-CDs (Figure 6a).

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Figure 6.

In the entity of AFB1 at different concentrations (25-200 ppm), (a) PL spectra of EA-CDs, (b) calibration curve of AFB1, (c) in the entity of AFB1 at different concentrations (25-200 ppm) absorbance spectrum of EA-CDs.

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Considering that both AFB1 and EA-CDs have fluorescence, we researched their reciprocal impacts on each other’s fluorescence properties. To evaluate the potential efficacy of EA-CDs in the detection of AFB1, the forster resonance energy transfer (FRET) of the reaction medium containing AFB1 and EA-CDs was determined by increasing the AFB1 concentrations at constant EA-CD concentrations (Figure 6a). The findings show that the presence of EA-CDs significantly increases the fluorescence of AFB1 (Figure 6a). All these results show that AFB1 can be sensitively detected in samples using EA-CDs. The results were also checked with UV-Vis spectra (Figure 6b). The calibration graph was plotted using PL spectra because the UV-Vis spectra showed small absorbance shifts as AFB1 concentration increased.

The observed fluorescence enhancement is redolent of the fact that EA-CDs may interact with AFB1 through mechanisms like the creation of fluorescent complexes or energy transfer. Fluorescence intensity was tested against AFB1 concentration, and a calibration curve was drawn (Figure 6c). The equation of the graph is y = -0.24356x + 635.46 (R² = 0.9908).

The emission spectrum of EA-CDs (centered at 450 nm) overlaps well with the absorption band at approximately 440–470 nm obtained upon addition of AFB1, providing strong spectral evidence of FRET. The findings conclusively demonstrate that the fluorescence intensity change observed in the EA-CDs-AFB1 system is primarily due to the FRET process, which is consistent with previous reports on CD-based biosensors [25].

In a specificity experiment for AFB1 exploration, folic acid, dopamine, ascorbic acid, cysteine, vitamin E, and urea were added to a solution including EA-CDs. AFB1 was set as the reference (100%) for fluorescence switch, and the relative responses of the other compounds were evaluated. Folic acid (21.67%), dopamine (42.91%), ascorbic acid (42.91%), cysteine (37.32%), vitamin E (29.41%), and urea (20.35%) indicated significantly lower fluorescence alteration compared to AFB1 (Figure 7). The difference between the results shows that EA-CDs have a significant specificity in AFB1 detection. Compared to the detection of selected compounds of metabolic importance, the PL emission values ​​obtained in the presence of AFB1 were considerably lower. This shows that EA-CDs are highly selective in AFB1 detection.

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Figure 7.

Specificity results of EA-CDs for AFB1 determination.

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Considering that AFB1 leads to improved fluorescence signals owing to its molecular construction, it therefore ensures more productive energy transfer among itself and EA-CDs. The low fluorescence results in other compounds may be due to the lack of similar interactions of AFB1 with the compounds. By doping the amino group of EA-CDs, they can be connected to the AFB1 structure with weak bonds (hydrogen bonding, van der Waals forces, etc.). The fluorescence quenching mechanism of EA-CDs makes them effective in detecting even very low amounts of AFB1. In this case, it facilitates the interaction with the three-dimensional structure of AFB1 and ensures the formation of stable intermediates in the binding. This is due to the strong interactions between the amine functional groups on the surface of EA-CDs and the AFB1 molecules, which increase the detection sensitivity of AFB1. There are also literature findings supporting our results [26]. This situation is promising for the use of EA-CDs in different samples.

AFB1 of a known amount (25 ppm) was added to some real samples and added to the EA-CDs medium, and the change in fluorescence emission intensities was measured (Figure 8). The change values ​​obtained were substituted into the calibration curve equation, and the amount of AFB1 was calculated. The % accuracy values ​​for the amount of AFB1 have been given in Table 1.

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Figure 8.

Fluorescence density measurements for the determination of the quantity of AFB1 in the real example of milk, corn, and peanut.

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As seen in Table 1, it is stated that EA-CDs have a very high accuracy rate, especially in peanut (99.2%) and corn (99.52%) samples. In the milk sample, the relatively low accuracy rate (82.16%) can be associated with the matrix factor; it can be thought that the liquid form or fluorescence elements in the sample may have affected it. Such a situation indicates that the method may need optimization in the milk matrix.

These findings show that the EA-CDs method has high accuracy rates, especially in peanut and corn samples. In the milk sample, the low accuracy rate (82.16%) can probably be associated with the matrix factor, or the sample being in liquid form, or fluorescence quenching. We achieved AFB1 detection in real samples ranging from 82.16% to 99.52%.

In general, the EA-CDs method can be seen as a valuable alternative for the determination of AFB1 in solid samples such as peanut and corn, and it may be useful to make improvements in the extraction or sample preparation steps to increase the accuracy rate in liquid samples. While EA-CDs demonstrated high sensitivity and selectivity in the detection of AFB1 under controlled conditions, it is conceivable that elements such as fat, protein, and pigments in the food sample could interfere with the determination. However, surface functionalization and optimization studies have shown that selectivity and stability have been improved [27].

To further support the selectivity of EA-CDs in the detection of AFB1 using their PL intensity properties, measurements were performed in the concentration range of 25 to 200 ppm. Statistical analysis was performed using one-way ANOVA followed by the Tukey post-hoc test. The findings confirmed that the PL intensity changes were significant (p < 0.01) compared to the other tested compounds. The quantitative and statistical analyses obtained strengthen the evidence that EA-CDs can reliably distinguish AFB1 from other biomolecules in complex food samples.

  While EA-CDs demonstrated high sensitivity and selectivity for AFB1 detection under controlled conditions, some potential limitations should be considered when applying the method to real food matrices. Complex food samples such as milk, nuts, and grains contain proteins, fats, carbohydrates, and natural pigments that can affect the fluorescence response of EA-CDs by causing quenching, scattering effects, or matrix-induced spectral shifts [28].

However, the accuracy achieved in this study for a milk sample with a high fat and protein content was 82.16%. While the percentage accuracy is not low, pre-purification and extraction procedures may be applied to such samples.

In this work, EA-CDs were synthesized from the EA plant using the pyrolysis method, and the aim was to investigate their usability in the detection of AFB1 in food samples. Since EA-CDS does not contain toxic material, does not require high energy, and is easy to apply, it can be used in AFB1 analysis in food samples. Particularly, CDs acquired from agrarian food refuse provision offer great benefits in terms of low cost and sustainability. When we look at the preceding studies, CDs were synthesized from diverse biological refuse, such as orange peel refuse, walnut shells, and corncobs, and they gave accomplished results, particularly in biomedical and biosensor implementation [29,30]. In SEM analysis, it was determined that the synthesized EA-CDs were spherical and 2.05 nm in size. In preceding work, it has been reported that CDs produced from agrarian waste products exhibit similar structural properties. For instance, it has been reported that CDs acquired from rice husk have similar amorphous construction and dimensions. The peaks obtained in XRD and XPS analyses of EA-CDs are consistent with graphite structures in the literature. This situation revealed that there were significant improvements in the optical and fluorescence properties of EA-CD prepared by doping with N, and this situation will easily enable its use as a biosensor [31,32]. The fluorescence spectra and UV-Vis spectra of EA-CDs show common peaks with the spectra seen in the literature. The fluorescence emissions they exhibited were related to different electronic transitions (π-π* and n-π*), and similar results are available in the literature [30].

The QY yield was determined as 54%, which also has the advantages of EA-CDs being environmentally friendly and non-toxic. In addition, N-doped CDs have increased their optical properties, which has increased their use as biosensors. In addition, their strong and decision-sensitive properties, even at basic pHs, are confirmed by literature data. For example, CDs acquired from food waste showed lofty fluorescence density in a basic environment. It was stated that this advantage could be used in biosensors. In a similar case, the decrease in fluorescence and red shift in acidic environments were clarified by the protonation of functional groups, and this condition was reported similarly in the literature [24,33]. The steadiness and selectivity of EA-CDs in the detection of AFB1 were attributed to the existence of functional groups, particularly nitrogen-doped (–OH, –NH₂, and C=O). Formerly reported CD-based biosensors in the literature also gave promising results in the detection of toxins like aflatoxin. For example, the achievement of CDs acquired from citrus peel in the detection of AFB1 is consistent with the conclusion of EA-CDs in this work. In preceding studies, it was accentuated that nitrogen doping provides CDs with a stronger interaction against toxins [34,35].

To further enhance the detection of AFB1 at low concentrations by EA-CDs, a combination of surface-specificity-enhancing functionalization (aptamer/antibody), signal amplification, robust sample preparation (solid-phase extraction (SPE)/immunoaffinity columns (IAC)), and matrix-matched calibration can be performed [36,37].