Nitrogen and sulfur co-doped carbon dots for ratiometric fluorometric determination of mercury ions

doi:10.21203/rs.3.rs-4949505/v1

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Nitrogen and sulfur co-doped carbon dots for ratiometric fluorometric determination of mercury ions

https://doi.org/10.21203/rs.3.rs-4949505/v1

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published 23 Oct, 2024

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Nitrogen and sulfur co-doped carbon dots (N,S-CDs) were prepared for dual-channel ratiometric fluorescence determination of mercury ions (Hg²⁺). The dual-emission N,S-CDs were synthesized using a simple one-pot hydrothermal treatment. When excited with visible light, N,S-CDs exhibited two emission peaks at 390 and 500 nm. Notably, the presence of Hg²⁺ caused a considerable decrease in the fluorescence of N,S-CDs at 500 nm, mainly due to the static quenching effect. In comparison, the fluorescence at 390 nm was almost unchanged. With a limit of detection (LOD) of 0.21 µM for Hg²⁺, the N,S-CDs were successfully applied to the unlabeled ratiometric fluorescence determination of Hg²⁺ in actual water samples with good recoveries (94.5–107.8%). In conclusion, this developed ratiometric fluorescent sensor provides a reliable, environmentally friendly, rapid, and efficient platform for detecting Hg²⁺ in environmental applications.

Carbon dots

Fluorescent probe

Ratiometric

Mercury ion detection

As a result of human activities, heavy metal contaminants in the environment have significantly surpassed acceptable limits, resulting in environmental deterioration and human health risks [1, 2]. Specifically, mercury ions (Hg²⁺), highly toxic representative heavy metal ions, are prevalent in water sources and can infiltrate the human body via the intricate food chain. Accumulation of excessive Hg²⁺ within the human system can inflict severe harm on vital organs, such as the brain, kidneys, central nervous system, and endocrine system, precipitating a spectrum of health conditions ranging from headaches and dizziness to numbness and tremors [3]. Thus, developing highly selective and sensitive detection methods for Hg²⁺ remains a pertinent and valuable pursuit.

The main Hg²⁺ detection methods studied include atomic absorption spectroscopy (AAS) [4], inductively coupled plasma–mass spectrometry (ICP–MS) [5], electrochemical methods [6–8], and fluorescent probe methods [9], which have high detection accuracy and good applicability. In particular, the analytical method based on fluorescent nanomaterials has advantages of a simple operation, low cost, inexpensive equipment, and strong anti-interference ability, and it has become common for detecting Hg²⁺ by researchers in recent years. For example, Chen et al. synthesized the fluorescent molecule coumarin–dithioacetal to achieve specific detection of Hg²⁺ by utilizing the specific recognition of Hg²⁺ by dithioacetal [10]. Liu et al. designed a far-infrared fluorescent probe, which used rhodamine derivatives as fluorescent groups and thiospironolactone as recognition sites to rapidly detect Hg²⁺ ions in cells and zebrafish [11]. However, most of these methods rely on single-emission fluorescence signals, and the accuracy of their results can be affected by changes in the external environment and instrumental conditions.

However, ratiometric fluorescence analysis eliminates the effects of changes in various factors on the measured fluorescence intensity [12, 13], such as the fluorescent probe concentration and instrumental conditions, and artificial artifacts, such as photobleaching, can be removed using the ratio of the two fluorescence peaks. Therefore, the ratio fluorescence analysis method has higher credibility and more practical significance in terms of results, which can compensate for the defects of the single fluorescence analysis method. Until now, several fluorescent nanomaterials, such as carbon quantum dots (CQDs) [14, 15], gold nanoparticles (Au NPs) [16, 17], and metal–organic skeleton nanomaterials (MOFs) [18, 19], have been developed for the ratiometric detection of Hg²⁺. However, these methods have unavoidable drawbacks, such as expensive raw materials and complicated synthesis processes for probes, and even contain toxic substances.

Carbon dots (CDs) are zero-dimensional novel nanomaterials with low fabrication costs, a simple synthesis method, excellent biocompatibility, low toxicity, and environmental friendliness compared with other nanomaterials [20, 21]. Therefore, this study proposes the synthesis of ratiometric fluorescent probes using nitrogen and sulfur co-doped carbon dots (N,S-CDs) for Hg²⁺ detection (Scheme 1). N,S-CDs were obtained using cysteine as the nitrogen and carbon sources, with two emission peaks at 390 and 500 nm under excitation of 310 and 410 nm. Due to the specific reaction between N,S-CDs and Hg²⁺, the fluorescence at 500 nm was quenched when N,S-CDs coexisted with Hg²⁺. At the same time, the fluorescence signal at 390 nm was almost unaffected, and the fluorescence remained stable. Thus, in this study, 500 nm was used as the response signal, and 390 nm was used as the built-in correction signal. The dual-emission peak fluorescence intensity ratios of N,S-CDs corresponding to different Hg²⁺ concentrations were used to achieve quantitative detection of Hg²⁺, and based on this, a rapid detection method was established for Hg²⁺ detection in environmental water samples.

Materials and instrumentation

L-cysteine, H₂O₂, Hg(NO₃)₂·H₂O, AlCl₃, CaCl₂, KCl, NaCl, CoCl₂, CuCl₂·2H₂O, FeCl₃·6H₂O, MnCl₂, MgCl₂, ZnCl₂, NaOH, and HCl were provided by Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). 4T-1cells and cell counting kit-8 (CCK8) were obtained from Beyotime Institute of Biotechnology (Shanghai, China). The other reagents used in the experiment were analytical reagent grade and were used without any further purification. Ultrapure water (18.2 MΩ/cm) was used for all experiments.

Fluorescence spectra were recorded using a Cary Eclipse fluorescence spectrophotometer (Agilent, USA). UV–vis absorption spectra were measured on a UV–2250 spectrophotometer (Shimadzu, Janpan). Transmission electron microscopy (TEM) images were obtained using a Talos F200 (Thermo Fisher, USA). Fourier transform infrared (FTIR) spectra were recorded using a NICOLET iS50 infrared spectrometer (Thermo Fisher, USA). X-ray diffraction was measured using a D8 Advance diffractometer (Bruker, Germany). Dynamic light scattering (DLS) measurements were performed using an Litesizer 500 (Anton Paar, Austria) at 25°C.

Preparation of nitrogen and sulfur co-doped carbon dots (N,S-CDs)

N,S-CDs were prepared according to reported studies with some modifications [22]. Briefly, 0.36 g of L-Cys, 300 µL of H₂O₂ (30 wt.%), and 15 mL of ultrapure water were added to a 50-mL sample vial with a lid (the pH of the mixture was 9.50). Subsequently, the vial was stirred at 90°C for 10 h, and the colorless mixed solution gradually turned bright yellow. Finally, the as-prepared N,S-CDs solution was purified using a dialysis membrane (molecular weight cutoff of 1000 Da) for 12 h with ultrapure water, and the product was collected and stored in a refrigerator at 4°C.

Detection of Hg²⁺

Detection of Hg^2 + by N,S-CDs probe was carried out in ultrapure water at 25°C. The sensing system was prepared with 20 µL of N,S-CDs and 20 µL of Hg²⁺ with different concentrations. The total volume was kept at 200 µL, and the final concentration of N,S-CDs was 25 µg/mL. After reacting at room temperature for 5 min, the fluorescence spectra were recorded at excitation wavelengths of 310 and 410 nm. The linear regression equation was obtained from the fluorescence ratio (F390/F500) versus the concentrations of Hg²⁺. Selectivity experiments were carried out using the same procedure. All experiments were performed in triplicate. Error bars showed the standard deviation (SD) of three parallel experiments. F390 and F500 denote the fluorescence intensity of the two emission peaks of N,S-CDs at 390 and 500 nm, respectively.

Detection of Hg²⁺ in actual samples

To investigate the usefulness of this sensor, we detected Hg²⁺ in lake water using the N,S-CDs sensing system. Tap water and lake water samples were obtained from laboratory and the artificial lake at Fujian Medical University, respectively. The water samples were filtered through a 0.22-µm microporous membrane and then centrifuged at 8000 rpm to remove particulate matter. To determine Hg²⁺ in the actual samples, 20 µL of N,S-CDs, 100 µL of water samples, and 20 µL of Hg²⁺ solution at various concentrations were added sequentially to a 0.6-mL plastic centrifuge tube (total volume 200 µL). The mixture was shaken and reacted for 5 min. Finally, the fluorescence spectra were collected at 310- and 410-nm excitation wavelengths.

Characterizations of N,S-CDs

First, the morphology of the synthesized N,S-CDs was investigated via transmission electron microscopy (TEM). Figure 1a shows a TEM image in which the N,S-CDs have a near-spherical appearance and good dispersion. The particle size of N,S-CDs was mainly distributed from 2.5 to 5.5 nm, and the average size was approximately 3.9 nm (Fig. 1a, insert). The HRTEM image in the upper right panel of Fig. 1a shows that the N,S-CDs have high crystallinity and distinguishable lattice stripes with a spacing of 0.22 nm, matching the lattice constants in the (100) plane of graphitic carbon [23], which is similar to previous studies on carbon dots [24].

FTIR and XPS were then carried out to analyze the structures and compositions of N,S-CDs. As illustrated in Fig. 1b, the absorption peak at 3435.4 cm^− 1 is attributed to the stretching vibrations of O–H and N–H, and the three cusp peaks at 1645.5, 1597.7, 1399.1 cm^− 1 are attributed to the stretching vibrations of C = O, C = C, and –C–N, respectively. The peak at 530.5 cm^− 1 is attributed to the bending vibration of C–S [22]. In addition, the XPS full spectrum in Fig. 1c shows that the N,S-CDs were mainly composed of C (283.5 eV), N (398.5 eV), O (530.4 eV), and S (162.7 eV), and the corresponding atomic contents were 70.5, 3.5, 22.9, and 3.1%, respectively. Furthermore, Fig. 1d shows the high-resolution XPS peaks for C 1s, which indicates the existence of C = O (288.0 eV), C–N (286.1 eV), and C–C (284.6 eV). The N 1s XPS spectrum indicates the presence of C–N–C (399.6 eV) (Fig. 1e). The high-resolution S 2p XPS spectrum shows a peak at 168.6 eV (Fig. 1f), which represents –C–SO₃– bonds [25]. The XPS results indicate that the synthesized CDs were successfully doped with N and S elements.

Based on the above results, the surface of N,S-CDs contains hydrophilic groups, such as –NH, –COOH, and –OH, which not only allow the N,S-CDs to be easily modified on the surface but also increase their hydrophilicity and stability in aqueous solution.

Optical properties of N,S-CDs

The optical properties of N,S-CDs were characterized by fluorescence and UV–vis spectroscopies. As shown in Fig. 2a, a strong excitation peak was present at 408 nm. The UV–vis spectrum of N,S-CDs shows two absorption peaks at 218 and 285 nm, which were ascribed to the π–π* transition of the aromatic sp² domains including C = C and C = N bonds and the n–π* transition of the C = O bond [26], respectively. The aqueous solution of N,S-CDs was light yellow under natural light and exhibited bright blue–green fluorescence under excitation at 365 nm (Fig. 2a, inset). Figure 2b shows the fluorescence spectra of N,S-CDs with excitation at 290–450 nm. When the excitation wavelength was gradually increased from 290 to 450 nm, two emission peaks at 390 and 500 nm were obtained at excitation wavelengths of 310 and 410 nm.

Due to their dual-wavelength emission properties, N,S-CDs can be developed as ratiometric probes. Figure 2c shows the fluorescence profiles of the N,S-CDs before and after adding Hg²⁺. Notably, the fluorescence at 390 nm remained stable, while the emission peak at 500 nm decreased when Hg²⁺ was added. In addition, Fig. 2d shows significant quenching of the green fluorescence of N,S-CDs with Hg²⁺, indicating that the probe could be used for Hg²⁺ detection. Thus, the stable fluorescence emission of 390 nm could be applied as a reference for the variable Hg²⁺-controlled fluorescence intensity of 500 nm as a ratiometric strategy.

Then, the effects of pH, ionic strength, and time on the fluorescence stability of N,S-CDs were evaluated. Fig. S1 shows a negligible change in the ratio of F390/F500 when the pH was between 4 and 12. However, the emission intensity of F390 remained stable, whereas that of F500 decreased as that the ratio increased when the pH was between 2 and 3. The pH responsiveness of the fluorescence of N,S-CDs likely results from the transition between C–NH₂ under acidic conditions and C = NH under alkaline conditions [22]. As shown in Fig. S2, the ratio was constant at different concentrations of NaCl solution. In addition, the F390/F500 ratio changed negligibly after 1 h of continuous laser irradiation, indicating a high resistance to photobleaching (Fig. S3). In addition, Fig. S4 shows that the fluorescence intensity of the N,S-CDs did not decrease considerably in irregular measurements over 60 days, indicating that these CDs have good fluorescence stability. These results indicate that the synthesized N,S-CDs are stable in an acidic environment, with excellent salt resistance, photobleaching resistance, and fluorescence stability, and are a luminescent material that has stable fluorescence performance, with the potential for practical application.

Furthermore, the biocompatibility of the prepared N,S-CDs was assessed, as shown in Fig. S5. After a 24-h incubation period with a series of concentrations of N,S-CDs, the viability of 4T-1 cells remained at an impressive 92%, even in the presence of 100 mg/mL N,S-CDs. This result highlighted the low cytotoxicity and excellent biocompatibility of N,S-CDs, suggesting promising application prospects in the biomedical field.

Optimization of experimental conditions

Several experimental conditions were optimized to improve the sensitivity of the ratiometric method for Hg²⁺ detection. As shown in Fig. S6, the F390/F500 ratio of the two emission peaks of the N,S-CDs did not change considerably when the pH was again kept between 4 and 12. The fluorescence ratio appeared to change after adding Hg²⁺ under the same conditions, and the ratio was maximized when the pH was 4. Therefore, a pH of 4 was selected as optimal for the ratiometric fluorescence detection of Hg²⁺. In addition, the reaction time was investigated. As shown in Fig. S7a, the fluorescence intensity at 390 nm remained constant, and the fluorescence at 500 nm decreased rapidly after 1 min of reaction. The F390/F500 ratio increased rapidly and reached an equilibrium minimum within 1 min of adding Hg²⁺ (Fig. S7b), suggesting rapid adsorption and quenching of Hg²⁺ by N,S-CDs.

Ratiometric Hg²⁺ sensing

Under optimized conditions, the quantitative detection of Hg²⁺ was assessed, revealing a distinctive pattern in Fig. 3a where the fluorescence intensity of F500 decreased upon adding Hg²⁺, while F390 remained stable. As shown in Fig. 3b, the F390/F500 ratio steadily increased with increasing Hg²⁺ concentration from 0 to 100 µM. Notably, Fig. 3c shows a strong linear correlation between the F390/F500 ratio and Hg²⁺ concentration within the 2–30 µM range, with a linear regression equation of y = 0.9242 + 0.004x and an R² value of 0.9954. The limit of detection (LOD) for Hg²⁺ using N,S-CDs, calculated based on the 3σ/k principle, was determined to be 0.21 µM, where σ represents the standard deviation from 12 blank solution repetitions, and k is the slope of the calibration curve. Compared to alternative Hg²⁺ detection methods (Table S1), the synthesized N,S-CDs exhibited commendable sensitivity and performance, further enhanced by their straightforward synthesis process and inexpensive raw materials. These results confirm that this method can effectively simplify the synthesis of ratiometric N,S-CDs with dual-channel detection capability.

Selectivity for Hg²⁺ detection

Specificity is one of the most pivotal performance indicators for evaluating sensing systems, as it gauges the probe’s ability to respond to a target analyte in a complex matrix selectively. In the context of fluorescent probes, the ideal scenario involves a precise and exclusive response to a specific metal ion. However, the reality can be more nuanced, with multiple ions potentially eliciting changes in the signal, necessitating rigorous testing to affirm the system's specificity. As demonstrated in Fig. 3d, a stark contrast emerged when an array of metal ions was introduced to the N,S-CDs. Among them, only Hg²⁺ triggered a pronounced augmentation in the fluorescence intensity ratio of 390 to 500 nm (F390/F500). This unambiguous enhancement served as compelling evidence of the ratiometric fluorescent probe’s specificity towards Hg²⁺, highlighting its ability to discern and respond solely to the targeted ion in a myriad of potential distractors. Moreover, the robustness of this detection method was validated by its resilience against interference from co-existing ions. Despite these potentially confounding factors, the fluorescence-based detection of Hg²⁺ persisted and was unaffected, underscoring the exceptional anti-interference capabilities of the established ratio detection strategy. This feature is particularly advantageous in real-world applications, where samples are often contaminated with multiple ions, making developing such highly specific and interference-resistant sensors all the more crucial.

Mechanism of Hg²⁺ detection

To verify the mechanism of the fluorescence quenching of N,S-CDs by Hg²⁺, we analyzed the UV absorption spectra, hydrodynamic diameters, and fluorescence lifetimes of N,S-CDs in the absence and presence of Hg²⁺. As shown in Fig. 4a, the fluorescence lifetimes of N,S-CDs in the presence and absence of Hg²⁺ were 2.47 ns and 1.99 ns, respectively, with little change, indicating that the effect of Hg²⁺ on the N,S-CDs was not due to dynamic fluorescence quenching [27, 28]. The absorption spectra showed that in the presence of Hg²⁺, the absorption peak of N,S-CDs at 285 nm shifted to a shoulder peak at 280 nm, probably due to the binding of N,S-CDs to Hg²⁺ and the transfer of electrons (Fig. 4b). To further analyze the fluorescent quenching mechanism, we measured the hydrodynamic diameter of the samples before and after Hg²⁺ detection using the N,S-CDs, and Fig. 4c shows that the average hydrodynamic diameter of N,S-CDs increased from 3.5 to 747.1 nm after adding Hg²⁺. This indicated that N,S-CDs were bound to Hg²⁺ and generated a non-fluorescent complex [29], which increased the ephemeral diameter. Based on those results, the detection of N,S-CDs on Hg²⁺ involved a static quenching mechanism, which is in line with our conclusions in this study.

Application in real samples

Tests were conducted on actual water samples to evaluate the practical applicability of N,S-CDs. As presented in Table S2, satisfactory recoveries ranging from 94.5 to 107.8% were achieved, accompanied by relative standard deviations (RSD) of less than 3.3%. These results demonstrated that the N,S-CDs-based ratiometric sensor provides a reliable and viable method for detecting Hg²⁺ in actual samples with high precision and excellent selectivity.

A ratiometric fluorescent sensor based on N,S-CDs was developed to detect Hg²⁺. Under optimized conditions, the probe showed a good linear relationship with Hg²⁺ in the concentration range of 2–30 µM with a correlation coefficient of R² = 0.9954 and the lowest detection limit of 0.21 µM. In addition, when detecting the heavy metal ions in actual lake water samples, the method showed high recoveries and good reproducibility, and it has the potential for rapid and reliable detection of heavy metal ions in environmental water samples.

Supplementary Information The online version contains supplementary material available at ......

Acknowledgments This work was supported by Natural Science Foundation of Fujian Province (No: 2023J05081, No: 2023J05082) and Fujian Provincial Department of Science and Technology University Industry-University Cooperation Project (No: 2023Y4014).

Author Contributions All authors contributed to the study conception and design. Material preparation, data collection, analysis and writing-original draft were performed by Fenglan Li, Liqin Lu and Yutong Wu. The writing-review & editing were performed by Liang Meng and Binling Zhu.The conceptualization, resources, supervision, and writing-review & editing were performed by Quanming Xu and Junyang Zhuang. All authors read and approved the final manuscript.

Data Availability No datasets were generated or analysed during the current study.

Competing Interests The authors declare no competing interests.

Ethical Approval Not applicable.

Consent to Participate Not applicable.

Consent to Publish Not applicable.

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Scheme figure is available in the Supplementary Files section.

No competing interests reported.

Scheme.jpg
Scheme 1 Schematic of the synthesis of N,S-CDs and their application for Hg²⁺ detection
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You are reading this latest preprint version

Nitrogen and sulfur co-doped carbon dots for ratiometric fluorometric determination of mercury ions

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Experimental section

Materials and instrumentation

Preparation of nitrogen and sulfur co-doped carbon dots (N,S-CDs)

Detection of Hg²⁺

Detection of Hg²⁺ in actual samples

Results and discussion

Characterizations of N,S-CDs

Optical properties of N,S-CDs

Optimization of experimental conditions

Ratiometric Hg²⁺ sensing

Selectivity for Hg²⁺ detection

Mechanism of Hg²⁺ detection

Application in real samples

Conclusion

Declarations

References

Scheme

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Nitrogen and sulfur co-doped carbon dots for ratiometric fluorometric determination of mercury ions

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Experimental section

Materials and instrumentation

Preparation of nitrogen and sulfur co-doped carbon dots (N,S-CDs)

Detection of Hg2+

Detection of Hg2+ in actual samples

Results and discussion

Characterizations of N,S-CDs

Optical properties of N,S-CDs

Optimization of experimental conditions

Ratiometric Hg2+ sensing

Selectivity for Hg2+ detection

Mechanism of Hg2+ detection

Application in real samples

Conclusion

Declarations

References

Scheme

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Detection of Hg²⁺

Detection of Hg²⁺ in actual samples

Ratiometric Hg²⁺ sensing

Selectivity for Hg²⁺ detection

Mechanism of Hg²⁺ detection