Highly fluorescent fish scale-derived carbon dots for quercetin sensing†

Chengzhi Xu^a, Binglu Wang^a, Jinyue Xing^a, Yanqiu Zhao^b, Lian Zhu^a, Juntao Zhang^a, Benmei Wei*^a and Haibo Wang*^c
aSchool of Chemistry and Environmental Engineering, Wuhan Polytechnic University, Wuhan, Hubei, China. E-mail: benmeiwei@whpu.edu.cn; Tel: +86-027-83943956
^bSchool of Food Science and Engineering, Wuhan Polytechnic University, Wuhan, Hubei, China
^cCollege of Life Science and Technology, Hubei Key Laboratory of Quality Control of Characteristic Fruits and Vegetables, Hubei Engineering University, Xiaogan, Hubei, China. E-mail: wanghaibo@whpu.edu.cn; Tel: +86-027-83956763

First published on 10th August 2024

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1 Introduction

Carbon dots (CDs) are a novel form of zero-dimensional carbon-based material. As a member of the carbon nanomaterial family, these exhibit good solubility, photostability, biocompatibility, low cost, and low toxicity, and have broad applications in areas such as biosensing and photocatalysis.^1–6 Top-down and bottom-up methods are the two main approaches popularly used for their synthesis. CDs can be prepared from a variety of precursors, including carbon-based materials, polymers, and biomass (depending on the preparation route).^7,8 Among them, biomass, as an abundantly available natural carbon source, has attracted a lot of attention due to its green, environmentally friendly, and renewable characteristics. Another significant advantage is that biomass can realize self-doping, i.e., the doping elements in CDs come from biomass itself, without the need to add any other reagents or additional modifications.⁹

The preparation of CDs with different fluorescence properties using biomass raw materials such as chitosan and silk fibroin has been studied previously.^10,11 However, these CDs often have a poor quantum yield. Further research on the functional modification of CDs has been carried out to improve their fluorescence properties. Element doping and surface modification are the primary techniques for improving or modifying the fluorescent properties of CDs. Element doping of CDs involves introducing different types of doping elements, such as nitrogen, sulfur, phosphorus, etc. to adjust the electronic properties and band gaps to obtain CDs with specific functions or better photoluminescent characteristics.^12,13 Surface modification mostly entails linking other chemical groups to modify the surface of CDs or introducing different functional groups to affect the energy levels of CDs, thereby changing or enhancing the light absorption and emission spectra of CDs, and yielding improved fluorescence properties.^14,15 Additionally, the optical properties of CDs can be modulated by coupling various specific polymers or biomolecules with CDs via electrostatic interactions, covalent bonds, or hydrogen bonds.^16,17

Fish scale waste is a low-cost and easily accessible nitrogen-containing biomass waste. Its main chemical components include collagen and hydroxyapatite, which are rich in carbon, hydrogen, oxygen, and nitrogen.¹⁸ Using fish scale waste as a raw material for preparing nitrogen-doped CDs offers the advantages of easy availability of precursors, simple reaction operations, and high value-added product generation, which has environmental and economic benefits. In previous work, CDs were prepared by employing fish scale as a raw material in a hydrothermal method under weak alkaline conditions to obtain a quantum yield of 6.04 ± 0.05%.¹⁹

This work aimed to improve the fluorescence properties of fish scale-derived CDs by evaluating the insertion of different compounds for doping or surface modification. High quantum yielding fish scale-derived CDs were created by screening the modifiers based on fluorescence quantum yield. The optical properties of high-quantum yield CDs were characterized by fluorescence and ultraviolet absorption spectroscopy. The microstructure and crystallinity of CDs were examined by transmission electron microscopy (TEM) and X-ray diffraction (XRD) analysis. X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) spectroscopy was used to decipher the intrinsic mechanism of the high quantum yield of CDs. Finally, the linear interval, detection limit, and recovery rate were studied for trace monitoring of quercetin using CDs. The overall idea of this work is shown in Fig. 1.

2 Materials and methods

2.1 Materials

Silver carp scales utilized in the present work were obtained from Wuhan Liangzihu Aquatic Processing Co., Ltd (Wuhan, China), and crushed using a frozen grinder (MM400, Retsch Co., Germany). Phytic acid, cysteine, tannic acid, catechin, caffeine, and glucose were obtained from Macklin Biochemical Co. Ltd (Shanghai, China). Glutathione, serine, leucine, and rutin were purchased from Innochem Science & Technology Co., Ltd (Beijing, China). Deionized water was used in all experiments. Other reagents were of analytical grade and procured from the Sinopharm Chemical Reagent Company, China.

2.2 Preparation of CDs

In separate beakers, 0.4 g of fish scale powder were precisely weighed. Then, a solution containing 0.03 mol L⁻¹ of various modified additives, including citric acid (CA), NaOH (SH), HCl (HA), H₂SO₄ (SA), ethylenediaminetetraacetic acid (EDTA), phosphoric acid (PA), phytic acid (IP6), glutathione (GSH), cysteine (Cys), and urea (urea), was added. The mixture was transferred to a hydrothermal reactor, sealed, and heated for 4 h at a constant temperature of 200 °C. After reaction completion, the mixture was collected and centrifuged at 12000 rpm for 10 min at 25 °C. The supernatant was then purified by dialysis for 72 h, using a dialysis bag with a molecular weight cutoff of 1000 Da. The resulting CDs were labeled as CDs-FS/X, where X denotes the abbreviation of the corresponding additive.

Furthermore, the following CDs were prepared as controls. Fish scale powder was added to pure water and phosphoric acid solution (as a pH adjuster), respectively, and subjected to the same hydrothermal reaction and purification to obtain CDs-FS, and CDs-FS/HA2, respectively. The Kjeldahl nitrogen analysis revealed a protein content of 57.7% in the fish scales, and an equivalent amount of collagen peptide was added to a citric acid solution and subjected to a hydrothermal reaction to obtain CDs-FSP/CA. Citric acid was added to water and subjected to a hydrothermal reaction to obtain CDs-CA. All the hydrothermal reactions were carried out in a DHG-9140A constant temperature incubator (Yiheng Scientific Instrument, China).

2.3 Characterization of CDs

The UV-vis absorption spectra of CDs in water were measured using a UV-2800 spectrophotometer (Unico Instrument, China). The Photoluminescence excitation and emission properties were investigated using an F98 spectrofluorometer (Lengguang Technology, China). During the measurements, the slit width was set to 10 nm, and the PMT voltage was maintained at 650 V. The time-correlated single-photon counting technique (FLS1000, Edinburgh Instruments, English) was utilized to determine time-resolved fluorescence behavior. The samples were excited with an EPL laser at 375 nm. The quantum yield of CDs was measured by comparing their absorbance and fluorescence intensity to those of quinine sulfate, a common fluorescent standard that has a quantum yield of 54% in 0.1 mol L⁻¹ H₂SO₄.

The crystal structure of CDs was characterized using an XRD-6000 diffractometer (Shimadzu Corporation, Japan) with CuKα radiation. The high-resolution structural images were acquired by using a field-emission high-resolution transmission electron microscope (JEM-2100F, Japan). The surface functional groups on the CDs were characterized using a NEXUS FTIR spectrometer (Thermo Fisher Scientific, USA). The elemental composition and valence states of CDs’ surfaces were analyzed using an ESCALAB 250XI XPS spectrometer (Thermo Fisher Scientific, USA).

2.4 Quercetin detection

For quercetin detection, CDs-FS/CA was added to quercetin solutions of varying concentrations and allowed to react for 5 min before measuring fluorescence emission spectra. The quercetin concentration in the mixed solution varied from 0–250 μmol L⁻¹. To determine the selectivity of CDs-FS/CA in detecting quercetin, the fluorescence emission spectra of CDs-FS/CA were measured in the presence of various micronutrients (glycine, serine, leucine, ascorbic acid, tannic acid, catechin, rutin, caffeine, glutathione), metal ions (K⁺, Mg²⁺, Mn²⁺, Cu²⁺, Co²⁺, Ca²⁺, Ba²⁺, Zn²⁺), and macronutrients (ethanol, glucose) solutions. The concentration of ethanol was 50 wt%, glucose was 5 wt%, and the concentration of the other components was 250 μmol L⁻¹.

The accuracy of CDs-FS/CA in detecting quercetin was further validated by examining the recovery rates of different concentrations (2.5, 12.5, and 25 μmol L⁻¹) of quercetin in actual beverage samples. Beer (ale, pilsner, and wheat beer) and tea (green tea, black tea) purchased from a local supermarket served as the actual beverage samples. The samples were only filtered and diluted before detection. Measurements were repeated a minimum of three times.

3 Results and discussion

3.1 Modification of carbon dots

Different compounds were introduced during the hydrothermal preparation process to increase the quantum yield of CDs derived from fish scales. The preliminary screening based on the fluorescence quantum yield (as shown in Fig. S1, ESI†), revealed that compared to other compounds, the incorporation of citric acid can greatly improve the quantum yield of the resulting CDs-FS/CA. Further optimization of the concentration of citric acid was conducted (Fig. S2, ESI†), and it was found that the highest fluorescence quantum yield (φ) of CDs-FS/CA (35.5 ± 0.1%) was obtained when the citric acid concentration was 0.1 mol L⁻¹. Therefore, the CDs-FS/CA prepared under these conditions were used for subsequent characterization analysis and applications.

3.2 Optical properties of modified CDs

Fig. 2 depicts the optical characteristics of CDs-FS/CA. As seen in the insets, the CDs-FS/CA disperses well in water, is transparent and colorless under visible light, but emits bright blue fluorescence under UV lamp excitation (365 nm). Fluorescence spectroscopy analysis revealed the optimal excitation peak of CDs-FS/CA at 350 nm, while the maximum fluorescence emission is at 411 nm (Fig. 2(A)). In the UV-vis absorption spectra, the CDs displayed a significant absorption peak in the range of less than 300 nm, which is generally attributed to as π–π* transitions within the sp²-hybridized graphitic core of CDs.^20,21

Further analysis of the luminescent properties of CDs-FS/CA at different excitation wavelengths was conducted. At excitation wavelengths ranging from 310 to 370 nm, the emission peak remained nearly constant at 411 nm. However, when the excitation wavelength was between 380 and 400 nm, the fluorescence emission of CDs-FS/CA exhibited a significant red shift (Fig. 2(B)). This indicates that the emission behavior of CDs-FS/CA is excitation wavelength-dependent. Moreover, these observations suggest that the fluorescence emission of CDs-FS/CA arises from at least two distinct emission states. This phenomenon could be attributed to surface modification or passivation, which induces different surface states leading to varied emission behaviors.^22,23

The photoluminescence decay of CDs-FS/CA was analyzed using time-correlated single-photon counting, and the data fit well to a biexponential decay model, as illustrated in Fig. S3 (ESI†). The photoluminescence lifetime (τ) was primarily governed by a long decay component of 9.83 ns (94%) with a minor contribution from a short decay component of 1.47 ns (6%). The weighted average lifetime was approximately 9.33 ns. Using the relationship of κ_r = φ/τ, the fluorescence radiative rate (κ_r) for CDs-FS/CA was determined to be 3.8 × 10⁷ s⁻¹. This high κ_r indicates a strong electronic transition probability in CDs-FS/CA, corroborating the proposed π–π* transition model.²⁴

Fluorescence stability is an important characteristic of CDs that is a prerequisite for their practical applications, as it enables better precision and reliability in chemical detection or biosensing.^25,26 Therefore, prepared CDs-FS/CA were evaluated for fluorescence stability under different testing conditions. To assess their photostability, CDs-FS/CA were continuously excited under a xenon lamp for 120 min (Fig. 2(C)). In an aqueous solution, under xenon lamp irradiation, CDs-FS/CA showed a slight decrease in fluorescence intensity. However, it still retained around 90% of its initial fluorescence intensity even after continuous irradiation for 120 min. In contrast, when dispersed in a 50% ethanol solution, the fluorescence intensity of CDs-FS/CA slightly increases after 20 minutes of xenon lamp irradiation and subsequently stabilizes. This phenomenon may be attributed to the combined effects of ethanol evaporation, which increases the concentration of carbon dots and thereby enhances fluorescence intensity, and the potential photobleaching caused by xenon lamp irradiation, which leads to fluorescence intensity decay.^24,27 These results indicate good photostability of CDs-FS/CA in both the aqueous and ethanol systems used for quercetin detection. Additionally, ionic strength presents another significant challenge for the practical application of CDs, as it can alter the state of surface groups on the CDs and affect the stability of the system. The effect of ion strength on the fluorescence intensity of CDs-FS/CA was investigated by adding NaCl solutions of different concentrations (Fig. 2(D)). It was found that there was almost no change in fluorescence intensity with increasing NaCl concentration. This demonstrates that CDs-FS/CA retains stable fluorescence properties even at high salt concentrations, indicating robust salt tolerance.

To sum up, CDs-FS/CA exhibits stable blue fluorescence emission properties. Furthermore, the mechanism of citric acid-modified CDs was investigated by analyzing the effect of its introduction on the structure of CDs.

3.3 Analysis of mechanism of citric acid modification

To explore the mechanism by which citric acid enhances the fluorescence emission of CDs, several control experiments were conducted as shown in Fig. 3(A). In the system where only citric acid solution was added, the quantum efficiency of CDs-CA was only 1.3 ± 0.1%, while in the system without citric acid, the quantum efficiency of CDs-FS was only 4.6 ± 0.1%. These were much lower than the obtained quantum efficiency of 35.5 ± 0.1%, when citric acid and fish scales were present together (CDs-FS/CA), indicating significant synergistic effects between citric acid and fish scales. Further, the quantum yield of CDs-HA2 was only 3.7 ± 0.3% when a hydrothermal reaction was conducted after adjusting the pH value of the reaction system to 0.1 using phosphoric acid, which was equivalent to the pH value of 0.1 mol L⁻¹ citric acid system. This suggests that the acidic environment introduced by citric acid was not the primary reason for the increased quantum yield of CDs.

Based on the above findings, attention was turned toward the inorganic component i.e., hydroxyapatite in fish scales. A control experiment was conducted without hydroxyapatite, wherein the protein content in silver carp scales (57.7%) was converted to collagen peptide equivalents. In the presence of citric acid, a quantum yield of 19.7 ± 0.3% was attained from CDs-FSP/CA. Although this was a significant increase as compared to the NaOH system (9.29%),²⁸ the quantum yield was still lower than that obtained in the presence of hydroxyapatite. These results suggest that introducing citric acid is an effective approach for increasing the quantum yield of CDs from natural protein sources, and inorganic components such as hydroxyapatite in fish scales also have a significant enhancing effect on the quantum yield of the resulting CDs.

The high fluorescence quantum yield of CDs-FS/CA might be attributed to changes in surface functional groups. The absorption bands of CDs were analyzed by UV-Vis spectroscopy, as shown in Fig. 3(B). The UV-Vis absorption spectrum of CDs-FS/CA displays two characteristic shoulder peaks at 275 nm and 335 nm. Interestingly, though CDs-FS and CDs-FSP/CA also have these two shoulder peaks, they are only prominent at one position, 275 nm for CDs-FS and 335 nm for CDs-FSP/CA. The absorption peak of CDs-CA is concentrated in the range below 250 nm. Generally, peaks at <300 nm reflect π–π* transitions corresponding to the conjugated CC of the carbon core.^21,29 The absorption peak at around 275 nm belongs to narrow bandgap absorption, benefiting from the contribution of conjugated domains.³⁰ The peaks at around 335 nm, assigned to n–π* transitions, may correspond to the influence of non-bonding orbitals from pyridine N and pyrrole N.³¹ The strong absorption of CDs-FS/CA at both positions suggests that its excellent fluorescence emission ability may not only be due to sp² C and different N-doped surface states but also due to their synergistic effects.³²

The crystal structure of CDs was analyzed using XRD, and all CDs exhibited broad and distinct diffraction peaks, as seen in Fig. 3(C). The diffraction angle 2θ of CDs-FS/CA, CDs-CA, and CDs-FSP/CA was around 21.4°, corresponding to the (002) interplanar spacing d of 0.41 nm. The CDs-FS had a diffraction angle 2θ of around 20°, corresponding to an interplanar spacing d of 0.44 nm. Compared to bulk graphite (about 0.34 nm), the CDs showed a larger spacing, which might be due to the rich O or N groups that weaken the aromatic interaction between the graphite layers.^33–35 All of these CDs exhibited amorphous carbon properties and a low degree of graphitization.

Comparing CDs-FS to the other three CDs revealed that it was more difficult for organic matter such as collagen in fish scales to be converted into CDs under neutral conditions than in an acidic environment containing citric acid, resulting in lower structural regularity and larger interplanar spacing. On the other hand, CDs-CA had a higher degree of graphitization, which can be explained by the fact that the raw material of CDs-CA was pure citric acid, which was more homogeneous relative to fish scales or fish scale peptides. The resulting CDs did not involve N doping, resulting in a more regular product structure.

The prepared CDs had a spherical shape and good dispersion without obvious aggregation, as observed in the HR-TEM images of Fig. 3(E). This might be related to the presence of hydrophilic functional groups on their surface. Based on the TEM images, the particle size distribution histogram was created by counting the particle size of the CDs (Fig. S4, ESI†). The distribution curve showed that the diameter of most CDs was below 3 nm. Compared with the other control CDs, CDs-FS/CA had the smallest average particle size (1.40 ± 0.30 nm) and a more concentrated distribution. CDs-FSP/CA had a similar but slightly larger and more scattered particle size distribution, while control CDs-FS and CDs-CA alone had larger particle sizes. High-magnification HR-TEM images revealed that these CDs had similar microstructures, consisting of a crystalline carbon core wrapped in an amorphous shell. The crystalline carbon cores display non-uniform lattice spacings of about 0.21–0.24 nm and 0.31 nm, indicating heterogeneity in the carbon core structure. These observation results were consistent with the results of the XRD analysis.

FTIR spectroscopy was used to examine the structural characteristics of the CDs, and the results are shown in Fig. 3(D). The FTIR spectra of all the CDs displayed a broad peak near 3400 cm⁻¹, which is related to the stretching vibration of –OH hydrogen bonds in CDs. The presence of hydroxyl functional groups not only makes the CDs hydrophilic but also boosts their stability and dispersion in water, making them more harmless in biochemical reactions. Compared to CDs-CA, the signals related to the stretching vibration of NH at 3270 cm⁻¹ were observed in CDs-FS, CDs-FS/CA, and CDs-FSP/CA. The additional peaks belonging to the stretching vibration of C–N were observed near 1300 cm⁻¹, indicating that nitrogen atoms were successfully doped in CDs. The bands of amide I (1660 cm⁻¹) and amide II (1580 cm⁻¹) were typical of protein FTIR spectra. In the infrared spectrum of CDs-FS, the amide II band disappeared, but the amide I band was still present. These results indicated that though protein structure was destroyed, the dehydration and carbonization of the protein were incomplete.³² The sharp peaks in the 1500–1700 cm⁻¹ range could be linked to the absorption of CC and –COOH vibrations, especially the signal peak at 1570 cm⁻¹ corresponded to the vibration of the benzene ring skeleton.

Furthermore, the signal peaks in the range of 1480–1350 cm⁻¹ in all CDs were mainly attributed to the C–H bending vibration in –CH₃/–CH₂–. The signal peaks corresponding to CDs-CA were the most obvious, while that corresponding to CDs-FS were the least obvious. A split peak appeared, which was closely related to their degree of carbonization. The obvious small peaks at 1245 and 1292 cm⁻¹ in CDs-CA, belonging to C–H, also confirmed that CDs-CA had the highest degree of carbonization.

The chemical structure of different CDs samples exhibited partial similarities, as reflected in the XPS full spectra (Fig. S5, ESI†). Three prominent peaks i.e., C1s, O1s, and N1s were apparent in CDs-FS/CA, CDs-FSP/CA, and CDs-FS, while CDs-CA had mainly two peaks: C1s and O1s. Additionally, a weak Ca2p peak, linked to hydroxyapatite in fish scales was visible in CDs-FS/CA and CDs-FS. Trace amounts of Ca play a pivotal role in the luminescent properties of CDs.³⁶ Alkali has been previously reported to significantly impact the surface electron state of CDs.^37,38

Further, the high-resolution spectra of the C1s, N1s, and O1s bands were deconvoluted (Fig. 4). The C1s spectra for C1s was fitted into peaks corresponding to C–C/CC (∼284.8 eV), C–N (∼285.9 eV), C–O (∼286.5 eV), and CO (∼287.6 eV) species (Fig. 4(A)).³⁹ The peak area comparison of different chemical states revealed that C–C/CC was the main form in CDs-CA. The other three chemical states (structural defects) were relatively few, indicating that CDs-CA had less heteroatom doping. Conversely, CDs-FS contained a large amount of doped C–N, C–O, and CO, validating the incomplete carbonization of CDs, consistent with the results of the analysis of the degree of carbonization of the first two CDs. CDs-FS/CA and CDs-FSP/CA had a relatively moderate degree of carbonization.

Except for CDs-CA, the N1s spectra of the other three CDs were deconvoluted into four distinct peaks assigned to pyridinic N (398.5 eV), amine N (399.7 eV), pyrrolic N (400.2 eV), and quaternary N (401.3 eV), respectively (Fig. 4(B)).^31,32 The specific analysis of the proportion of different types of N revealed that CDs-FS contained a higher proportion of amine N, which could be due to its lower degree of carbonization. The greater pyrrolic N content of CDs-FS/CA and CDs-FSP/CA improves charge mobility and donor–acceptor properties of CDs, enhancing their quantum yield. The O1s spectra (Fig. 4(C)) were also deconvoluted into two peaks at 531.1 eV and 532.9 eV, which correspond to the presence of CO and C–OH/C–O–C respectively.³² The CDs-FS/CA and CDs-FSP/CA contained more C–OH/C–O–C, while CDs-FS and CDs-CA had higher levels of oxygen in the form of CO. The relatively low proportion of C–OH/C–O–C in CDs-CA may be linked to its higher degree of carbonization and more regular structure, while the higher proportion of CO in CDs-FS mainly comes from undestroyed amide bonds.

Based on the above analysis, the introduction of citric acid and the participation of hydroxyapatite were pivotal drivers for the high quantum yield of CDs derived from fish scales. Structural analysis of the CDs reveals that CDs-FS/CA possess a moderate degree of graphitization, a smaller minimum average particle size, concentrated distribution, as well as a higher proportion of pyrrolic N. The synergistic effects of surface states of sp² C and varied N doping likely play a key role in their excellent fluorescence properties. However, further investigation is required to elucidate the specific effects of citric acid on the surface energy levels and surface charge transfer of biomass-derived CDs. This will provide important insights for the regulation of surface states and the optimization of performance in biomass-derived CDs.

3.4 Trace detection of quercetin

Polyphenols have attracted the attention of many researchers as a result of people's pursuit of a high-quality life and a nutritious diet. Quercetin is one of the most abundant polyphenolic compounds found in foods such as fruits, wine, beer, and tea. Its characteristics constitute anti-tumor, antiviral activity, anti-mutagenesis, cardiovascular protection, and cataract prevention.^40,41 Therefore, quercetin determination has great relevance in biochemistry, clinical medicine, and natural chemistry. Traditional quercetin fluorescent probes suffer from shortcomings such as low sensitivity, and interference from other flavonoids. However, the most important factor which limits their application in biology is that commonly used fluorescent molecules and quantum dots are cytotoxic.⁴² Therefore, developing a simple and low-toxicity optical sensor material with high selectivity and sensitivity is of great significance. Hence, the prepared CDs-FS/CA were used to detect quercetin in food samples.

Fig. 5(A)–(C) displays the fluorescence spectra of CDs-FS/CA solutions with varying quercetin concentrations, after excitation with a 350 nm wavelength. The fluorescence intensity gradually decreased with the increase in quercetin concentration. In the 0–50 μmol L⁻¹ range, a correlation coefficient (R²) of about 0.992 and a linear equation of y = 1.19x + 4.43 denoted a strong linear relationship between the quenching efficiency and quercetin concentration. The detection limit (LOD) was 3.8 nmol L⁻¹. The detection limit was calculated using 3σ/k, where σ is the standard deviation of blank measurement, and k is the slope obtained after linear fitting. Furthermore, the limit of quantification (LOQ) was calculated using the equation LOQ = 3.3 × LOD, resulting in a value of 12.4 nmol L⁻¹. Using a quercetin concentration of 12.4 nmol L⁻¹, seven replicate experiments were conducted. The true method detection limit (MDL) and method quantification limit (MQL) were determined to be 3.0 nmol L⁻¹ and 9.9 nmol L⁻¹, respectively (as detailed in Table S1, ESI†).

Table 1 depicts the relevance of the present study method for detecting quercetin in comparison to previous literature reports on quercetin determination. The present approach enabled a higher detection limit and the improvement of LOD in this fluorescent probe is of great significance.

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To further validate the fluorescent system's selectivity, various micronutrients, metal ions, and macronutrients were studied for their fluorescence quenching effect on CDs-FS/CA. These included glycine, serine, leucine, ascorbic acid, tannic acid, catechin, rutin, caffeine, glutathione, quercetin, K⁺, Mg²⁺, Mn²⁺, Cu²⁺, Co²⁺, Ca²⁺, Ba²⁺, Zn²⁺, ethanol, and glucose. Among them, ethanol concentration was 50 wt%, glucose concentration was 5 wt%, and all other components were at a concentration of 250 μmol L⁻¹. As shown in Fig. 5(D), the fluorescence quenching of K⁺, Mg²⁺, Mn²⁺, Ca²⁺, Ba²⁺, Zn²⁺, glycine, serine, leucine, ascorbic acid, caffeine, glutathione, ethanol, and glucose was insignificant, but quenching was observed for Cu²⁺, Co²⁺, tannic acid, catechin, and rutin. It is worth noting that tannic acid and catechin are both polyphenolic substances, while rutin, like quercetin, belongs to the flavonoid class. These three compounds share structural similarities with quercetin. Although they exhibit fluorescence quenching effects, the extent of quenching is less than 30%. The quenching of quercetin, on the other hand, was more than 95%, indicating that CDs-FS/CA has a high recognition ability for quercetin. The strong interaction between quercetin and the surface amino groups of CDs-FS/CA is likely the driving mechanism behind the specific fluorescence quenching effect observed. The surface of CDs-FS/CA is rich in basic groups such as amides and amines. The most acidic proton in quercetin is located on the hydroxyl group of the C ring (3-hydroxychromone site), with a pK_a of 6.74. Therefore, quercetin is likely to anchor onto the surface of CDs-FS/CA through electrostatic interactions between the 3-hydroxyl group of quercetin and the basic groups on CDs-FS/CA, further promoting the dissociation of the 3-hydroxyl group and quenching the fluorescence of CDs-FS/CA.⁴⁵

A recovery test is an important tool for evaluating the accuracy of detection methods in practical applications. In this study, quercetin was added at concentrations of 2.5, 12.5, and 25.0 μmol L⁻¹ to three types of beer samples (Ale, Pilsner, and Wheat) and two types of tea drink samples (Green Tea and Black Tea), and then subjected to recovery test analysis. As shown in Table 2, the recovery rates ranged from 87.2% to 109.4%, demonstrating that quercetin detection using CDs-FS/CA has high accuracy. Therefore, this method can be used to determine the quercetin content in beer, tea, and other real-world samples. Furthermore, the approach offers benefits such as environmentally friendly reagents, no need for complex sample pretreatment, no solvent extraction steps, and feasible analysis after simple filtration and dilution steps. These advantages make it a promising method for practical detection in various fields.

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To investigate the interaction mechanism between CDs-FS/CA and quercetin, the effect of quercetin introduction on the UV-visible absorption spectra of CDs was examined, as shown in Fig. 6(A). Upon interaction with quercetin, the absorbance of the UV-visible absorption spectra of CDs-FS/CA decreased significantly, indicating that CDs-FS/CA may interact with quercetin to form a non-fluorescent ground-state complex, thereby reducing the UV absorption and fluorescence intensity of CDs. This suggests the presence of static quenching in the CDs-FS/CA and quercetin interaction system.^48,49 Moreover, quercetin exhibits characteristic absorption peaks around 255 nm and 375 nm, with the 375 nm peak situated between the excitation wavelength (350 nm) and emission wavelength (411 nm) of CDs-FS/CA. The significant decrease in absorbance at 375 nm upon interaction with CDs-FS/CA implies the presence of a post-filter effect.

To further elucidate the quenching mechanism of quercetin on CDs-FS/CA, temperature-dependent experiments and fluorescence lifetime analyses of the carbon dots were conducted. As shown in Fig. 6(B), the quenching effect of quercetin increases significantly with rising temperature, indicating the presence of dynamic quenching. The reduction in the fluorescence lifetime of the carbon dots further supports the existence of dynamic quenching within the system (Fig. 6(C)).^50,51 Therefore, it can be inferred that both static and dynamic quenching are involved in the quercetin-induced quenching of CDs-FS/CA.

4 Conclusion

Fish scales were used as a precursor for the one-step hydrothermal synthesis of CDs. To improve the quantum yield of CDs, different modification additives were introduced during the hydrothermal process It was found that introducing citric acid could significantly improve the quantum yield, up to 35.5 ± 0.1% for CDs-FS/CA. Fluorescence properties and structural performance of CDs-FS/CA were characterized. The CDs-FS/CA exhibited bright blue fluorescence under UV irradiation, good dispersion, photostability, salt resistance, and emission behavior independent of excitation wavelength. Further comparative studies revealed that the inclusion of citric acid and participation of hydroxyapatite in fish scales were significant contributors to the high quantum yield of CDs-FS/CA. The prepared CDs-FS/CA had moderate graphitization, a smaller average particle size with a more concentrated distribution, and a higher proportion of pyrrole N. They exhibited good fluorescence performance due to the synergistic effect of surface state sp² C and varying N doping., A strong linear relationship was observed in the 0–50 μmol L⁻¹ range using CDs-FS/CA for the trace detection of quercetin, with a detection limit of 3.8 nmol L⁻¹. Moreover, this approach demonstrated a good recovery rate in the quercetin detection from actual beverage samples, indicating its promising application potential in various sectors.

Ethical approval

This research did not involve human or animal samples.

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of 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.

Acknowledgements

The authors would like to thank all the reviewers who participated in the review. This research was supported by the National Natural Science Foundation of China (no. 22178277, 22378320), the Knowledge Innovation Program of Wuhan-Basi Research (no. 2023020201010148).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4nj02578d

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