Trends Sci. 2025; 22(9): 10340

Introduction

Food safety is a global concern, particularly azo dyes such as Amaranth (E123) in food and beverage products. Although effective as a colorant, Amaranth can cause liver damage and bladder cancer [1]. The limitations of current detection methods inhibit the monitoring of Amaranth levels in food products, thus potentially endangering consumer health. Various methods have been developed to detect Amaranth, such as absorption spectroscopy, chromatography, and electrochemical methods. However, these methods have limitations in terms of low sensitivity and complicated preparation [2].

Carbon Dots (CD) have emerged as a promising alternative as they are zero-dimensional carbon nanoparticles under 10 nm in size and are capable of fluorescence in various colors [3]. The fluorescence ability is related to the structure of the carbon core surrounded by functional groups, giving it unique properties such as high water solubility and good response to the environment [4]. To improve the performance of CD, many doping techniques have been developed.

The co-doping of nitrogen and sulfur on CD has the potential to improve the fluorescence performance, where the enhancement mechanism depends on the electrons trapped in the N state and the modification of the electronic structure of CD [5]. Meanwhile, the addition of sulfur increases the density of states so that the excited electrons can increase the fluorescence intensity [6]. In recent developments, N, S-CD have been successfully used as fluorescence sensors for azo dyes including amaranth, with a ratiometric approach that provides the advantage of using the ratio of 2 emissions at different wavelengths, thus providing internal calibration and improving detection accuracy [7].

Although N, S-CD sensors have shown potential, the development of sensors with better performance is still needed. The biomass source used for CD synthesis greatly affects its characteristics and performance [8]. Jengkol peel is a biomass waste that contains a lot of nitrogenous heterocyclic organic compounds [9,10] has great potential for N, S-CD synthesis but has never been explored. The high nitrogen content in the jengkol peel is expected to increase the fluorescence intensity and stability due to its ability to bind the CD core. In addition, CD synthesized from biomass generally have better optical properties and are environmentally friendly compared to semiconductor quantum dots [11].

This study proposes the development of an N, S-CD based ratiometric fluorescence sensor using jengkol peel as a biomass source. This approach not only offers sensor performance content in the jengkol peel but is also in line with the principle of green chemistry, as it utilizes biomass waste for detecting amaranth.

Materials and methods

Material

Jengkol peel was obtained from Sumatera Utara Province, Indonesia, Aquadest, Deionisasi water, Urea (CH₄N₂O), natrium tiosulfat (Na₂S₂O₃), amaranth (95 %), nickel (II) nitrate hexahydrate (Ni(NO₃)₂), mercury(II) nitrate (Hg(NO₃)₂, copper (II) nitrate (Cu(NO₃)₂, cobalt (II) nitrate (Co(NO₃)₂, iron (II) nitrate (Fe(NO₃)₂, manganese (II) nitrate (Mn(NO₃)₂, zinc (II) nitrate (Zn(NO₃)₂, natrium chloride (NaCl), ascorbic acid, caffein, citric acid, glucose, tartazine, rhodamine blue were acquired from Sigma-Aldrich. All chemical com-pounds were utilized unpurified as received and belonged to the grade of analytical reagents.

Synthesis of carbon dots (CD)

The Jengkol peel is dried out and then powdered. The 10 g of jengkol peel powder was dissolved in 50 mL of deionized water. Subsequently, the liquid was poured into a 100 mL Teflon autoclave and heated at 220 °C for 7 h. The Whatman filter paper was used to filter the resulting dispersion and centrifuged at 10,000 rpm for 15 min to yield a brown solution. The obtained supernatant was then purified through a dialysis process using a membrane with a molecular weight cut-off of 10,000 DA for 24 h. The final product from this process was named CD.

Synthesis of nitrogen and sulphur co-doped carbon dots (N, S-CD)

Firstly, 10 g of jengkol peel powder, 5 g of urea, and 5 g of sodium thiosulfate were weighed and mixed in 50 mL of deionized water. Subsequently, the mixture was poured into a 100 mL Teflon autoclave and heated at 220 °C for 7 h. The Whatman filter paper was used to filter the resulting dispersion and centrifuged at 10,000 rpm for 15 min to yield a brownish-yellow solution. The obtained supernatant was then purified through a dialysis process using a membrane with a molecular weight cut-off of 10,000 DA for 24 h. The final product from this process was named N, S-CD.

The fluorescence measurements of amaranth dye by N, S-CD

N, S-CD were synthesized as sensor probes to detect amaranth dye. The selectivity and sensitivity of amaranth dye were investigated based on the fluorescence quenching efficiency at 370 nm. Experimental solutions were prepared as different control substances, such as amaranth (95 %), sodium chloride (NaCl), ascorbic acid, caffeine, citric acid, glucose, tartazine, rhodamine blue, and 7 different metal ions (Ni²⁺, Hg²⁺, Fe²⁺, Mn²⁺, Cu²⁺, Co²⁺ and Zn²⁺). The N, S-CD solution (2 mL) was added with each at a concentration of 100 μM. The sensitivity of amaranth dye was examined further by incorporating amaranth concentration from (2.5 - 20 μM) into 2 mL of N, S-CD solution. Subsequently, fluorescence emission spectra were obtained, and the intensity of the emission peak was noted in the ambient.

N, S-CD on amaranth dye was tested on actual samples such as tomato juice and strawberry syrup. Samples of 15 mL were centrifuged for 30 min at 3,500 rpm. The supernatant was filtered using a 0.22 μM filtration membrane and diluted ten times with deionized water. Then, 2 mL of the resulting filtrate was spiked with several concentrations of amaranth and stirred for 10 min after adding 2 mL of N, S-CD solution. The actual concentration of amaranth was obtained through the measured fluorescence intensity.

Quantum yield measurement

Quantum yield (QY) investigation on CD and N, S-CD utilizing the equation that follows (1), Quinine sulfate dissolved in 0.1 M H₂SO₄ (QY = 54 % and η = 1.33) at 360 nm, used as reference [12].

where the quantum of yield represents QY, it relates to the brightness of the emission fluorescence. A signifies the absorbance t measured under the excitation wavelength, and η represents the solvent refractive index. C dan S, respectively for N, S-CD, and quinine sulfate. The photoluminescence spectrum was acquired with a Fluorescence Spectrometer

Results and discussion

Morphology, structure, and optical properties of CD and N, S-CD

Carbon dots were synthesized rapidly and environmentally friendly through the hydrothermal method. That treatment can dehydrate and decarboxylate the jengkol peel precursor to generate sp²/sp³ hybridization domains and various surface states on N, S-CD. Characterization results from TEM analysis in Figures 1(a) and 1(b) show that CD and N, S-CD have a spherical particle morphology without aggregation, with an average size of 4.47 nm for CD and 7.41 nm for N, S-CD based on measurements of 100 particles. The increase in particle size in N, S-CD compared to CD indicates the successful doping of nitrogen and sulfur which enhances the nucleation activity and facilitates the formation of larger particles [13]. The findings support previous research showing N,S co-doping increases CD particle size, which is favorable for sensor applications because it provides a larger surface area for interaction with analytes [14].

XRD analysis (Figure 1(c)) confirmed the amorphous structure of CD with a broad peak (002) at 2θ = 24.02 ° [15], while N, S-CD showed increased crystallinity characterized by a sharp diffraction peak (002) at 2θ = 28.41 ° (FWHM = 0.14210) [16] and a lattice spacing of about 0.34 nm. The increase in crystallinity indicates that N, S co-doping not only affects the particle size but also induces the formation of sp² which contributes to the enhancement of graphitization and polycrystalline nature of carbon. These structural characteristics play an important role in improving the fluorescence properties of N, S-CD, This contrasts with earlier studies where CD exhibited amorphous structures [17].

XPS analysis revealed the successful modification of the CD structure through the doping process. CD in Figure 2(a), shows the content of C1s and O1s (53.8 and 46.2 %) with characteristic peaks at 284.58 and 531.87 eV, as well as 3 main bond types at C1s resolution: C-C/C=C (284.58 eV), C=O (286.50 eV) [18]. Figure 2(b) reveals that N, S doping led to the incorporation of nitrogen (9 %) and sulphur (16.8 %), confirmed by absorption peaks at 163.67 eV (S2p) [19], 284.67 eV (C1s), 398.67 eV (N1s), and 531.67 eV (O1s). Resolution of C1s on N, S-CD showed the formation of new bonds at 284.67 eV (C=C/C-C), 285.4 eV (C-N/C-S), 287.3 eV (C=O), and 291 eV (C(O)-O), with an N1s peak at 398.67 eV from N pyrrolate and N pyridine confirming the formation of amino groups [20], while the S2p bond energy value indicates the presence of C-S-C bonds [21]. The high doping percentages demonstrate jengkol peel effectiveness as a nitrogen-rich precursor, resulting in N, S-CD with uniformly distributed dopants.

Figure 1 TEM images and inset of the size distribution of CD (a) and N, S-CD (b), and XRD spectra of CD and N, S-CD (c).

Figure 2 XPS spectra and high-resolution inset of C1s of CD (a) and N, S-CD (b).

Figure 3 FTIR spectra of CD and N, S-CD.

FTIR spectra of N, S-CD, and CD (Figure 3) showed absorption bands at 3,231, 3,369 and 1,647 cm⁻¹ (OH, N-H, C-H and C=H/C=C groups) [21]. These hydrophilic groups facilitate water dispersibility and create an optimal conjugation system for fluorescent emission [22]. The N, S-CD spectrum shows peaks at 1,412, 1,162 and 1,001 cm⁻¹, corresponding to C-N, C-S, and C-O-C vibrations, which confirms nitrogen and sulfur incorporation into the sp² carbon skeleton [23].

UV-Vis spectroscopy (Figures 4(a) and 4(b)) reveals absorption band edges at 293 and 294 nm for CD and N, S-CD, respectively, attributed to π-π* transitions of C=C bonds. N, S-CD exhibits a distinct absorption at 348 nm due to n-π* transitions of CN, C=O, and CS bonds, where surface modification creates a p-conjugated structure leading to a red shift in fluorescence emission [24]. PL characterization (Figures 4(a) and 4(b)) reveals emission peaks at 462 nm (CD) and 515 nm (N, S-CD) with excitation wavelengths between 330 - 390 nm (Figure 4(c)). The redshift in emission wavelength accompanied by decreased PL intensity confirms N, S-CD excitation-dependent fluorescence properties [25].

Figure 4 UV-visible spectra and PL spectra of (a) CD (b) N, S-CD. (c) excited N, S-CD (λex) with different wavelengths.

Excitation at 370 nm produces maximum intensity for both CD samples, where the N, S-CD surface functional groups facilitate the π-π* electronic transition at the C=C bond [26]. This transition process leads to emission variations from different surface states, primarily through interactions between reactive oxygen/nitrogen functional groups and the N, S-CD carbon skeleton, modifying the energy band gap. The functional groups act as energy traps on N, S-CD, influencing PL emission characteristics, evidenced by increased quantum efficiency from 4.5 % (CD) to 20.5 % (N, S-CD) through band gap narrowing. These changes confirm successful optoelectronic property modification via doping.

Optimization of N, S-CD sensing parameters

Parameter optimization for amaranth sensing by N, S-CD was conducted through analysis of pH, ionic stability, luminescence performance, and photostability. Using a 100 μM amaranth solution, quenching efficiency was measured via the F0/F ratio. pH analysis (range 2 - 9) showed increasing fluorescence intensity from pH 2 to pH 7 (Figure 5(a)), indicating complex interactions between amaranth and N, S-CD through protonation and deprotonation.

In acidic conditions, protonation of oxygen groups on the surface of N, S-CD induces changes in energy levels that contribute to an increase in fluorescence intensity [27]. In contrast, at alkaline pH (8 - 9), deprotonation of functional groups resulted in a decrease in fluorescence response, confirming the critical role of proton balance in the sensing mechanism. The establishment of pH 7 as the optimal condition reflects the optimal balance between protonation and deprotonation for sensor-analyte interaction.

The analysis of N, S-CD storage stability in aqueous solution (Figure 5(b)) demonstrates excellent material durability, with fluorescence intensity maintained consistently throughout a 30-day period. This long-term stability indicates the strength of covalent bonds within the N, S-CD structure, which plays a crucial role in maintaining sensor integrity during storage.

Fluorescence response of N, S-CD to amaranth dye

Evaluation of the selectivity of N, S-CD (Figures 6(a) and 6(b)) towards various interference components including NaCl, ascorbic acid, caffeine, citric acid, glucose, tartrazine, rhodamine blue, and 7 metal ions (Ni²⁺, Hg²⁺, Fe²⁺, Mn²⁺, Cu²⁺, Co²⁺ and Zn²⁺) revealed high selectivity towards amaranth. This selectivity occurs through the formation of hydrogen bonds between the hydroxyl/carboxylate groups of amaranth and the N/S groups on the N, S-CD surface, where the co-doped charge distribution favors electrostatic interactions and complex stability [28]. As a result of the interaction, there is a decrease in fluorescence intensity at 515 nm and an increase at 646 nm as the amaranth concentration increases (Figure 6(c)), indicating a resonance energy transfer mechanism that allows the development of a ratiometric detection system (F515/F646) in the range of 2.5 - 20 μM.

The change in intensity ratio is inversely proportional to amaranth concentration, marked by the color transition from cyan to yellow under UV excitation (Figure 6(e)), resulting in a detection limit of 7.5 nM with high linearity (R² = 0.99) can be shown in Figure 6(d). The limit of detection (LOD) was estimated using:

where σ represents the standard deviation of the blank (n = 3) and s is the slope of the linear curve.

This ratiometric method at 2 different wavelengths eliminates matrix interference and measurement fluctuations, providing better accuracy and reliability than conventional single intensity-based fluorescence methods [29]. The sensor performance comparison in Table 1 confirms the effectiveness of the N, S-CD surface modification for azo dye detection over other carbon dot sensors.

Table 1 Literature review comparing the proposed research with other reports on amaranth and detection methods

Table 2 Investigation of amaranth in actual sample.

Analysis of fluorescence quenching mechanism

Analysis of the UV-Vis, emission, and excitation spectra of N, S-CD indicates fluorescence quenching by amaranth through FRET (Förster Resonance Energy Transfer) [30] . This is supported by the overlap between the emission spectrum of N, S-CD and the absorption spectrum of amaranth (Figure 6(f)), along with amaranth photoluminescence emission at 628 nm, which confirms that the emission at 646 nm originates from amaranth excited through FRET. In this mechanism, N, S-CD acts as an energy donor (intensity at 515 nm decreases), while amaranth as an acceptor produces an induced emission at ~650 nm whose intensity increases with amaranth concentration, demonstrating ratiometric characteristics with the F515/F646 ratio inversely proportional to amaranth concentration, confirming efficient resonance energy transfer.

Figure 6 (a) and (b) fluorescence in various substances. (c) Fluorescence intensity decreases the spectrum of N, S-CD by adding amaranth concentration. (d) Relationship between F515/F646 and amaranth concentration. (e) Color emission shift from blue to yellow upon amaranth detection (f) Overlap scheme of N, S-CD spectrum.

Analysis by actual sample

Validation of the amaranth detection method in complex matrices using strawberry syrup and tomato juice as model food samples (Table 2) was carried out through the standard addition method at 3 concentrations (1, 3 and 5 μM). Testing in strawberry syrup resulted in 94 - 98 % amaranth recovery (RSD < 1.5 %), while in tomato juice it reached 94 - 96 % (RSD < 2.5 %). The high recovery rate and low RSD values confirm the selectivity and reproducibility of the method in detecting amaranth in beverage matrices.

Conclusions

Ratiometric fluorescence probes were fabricated through surface modification of carbon dots using nitrogen and sulfur co-doping from the jengkol peel precursor via a 1-pot hydrothermal method with urea and sodium thiosulfate as dopants. The material exhibits spherical morphology with a 7.41 nm diameter and enhanced crystalline structure through N, S co-doping, achieving a 20.5 % quantum yield. The sensor produces a ratiometric response through interactions between N, S-CD surface functional groups, and amaranth via electron transfer mechanisms at dual wavelengths (emission/excitation 515/370 nm), with a linear detection range of 2.5 - 20 μM and 7.5 nM LOD. Its effective performance in real sample applications demonstrates the potential for developing biomass-based sensors for food safety analysis.

Acknowledgements

The authors gratefully acknowledge the support of Universitas Sumatera Utara (USU) in advancing the World Class University program. This work was supported by the Ministry of Research and Technology of the Republic of Indonesia with contract number 60/UN5.2.3.1/PPM/KP-WCU/2022.

Declaration of Generative AI in Scientific Writing

The authors acknowledge that generative AI (such as Quillbot and ChatGPT) was used in the preparation of this manuscript. The use of AI was for editing and grammar correction. No content was generated, nor was data interpreted using AI. All content and conclusions in this paper are based on our contributions and analysis.

CRediT Author Statement

Marpongahtun: Conceptualization, Methodology, Supervision, Validation, Funding acquisition, and Writing –original draft.
Amir Hamzah: Formal analysis, Validation, Supervision and Investigation.

Agus Rimus Liandi: Formal analysis, Supervision and Investigation.

Amru Daulay: Formal analysis, Supervision, and Investigation.

Salmiati: Formal analysis, Investigation, Validation, and Visualization.

Aniza Salviana Prayugo: Methodology, Data curation and Writing –original draft

Ririn Annisa: Methodology, Data curation and Writing –original draft

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