N,S-self-doped carbon quantum dots from fungus fibers for sensing tetracyclines and for bioimaging cancer cells

Materials Science & Engineering C 105 (2019) 110132

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N,S-self-doped carbon quantum dots from fungus fibers for sensing tetracyclines and for bioimaging cancer cells

T

Cai Shia,1, Houjuan Qia,1, Rongxiu Maa, Zhe Suna, Lidong Xiaoa, Guangbiao Weia, ⁎ Zhanhua Huanga, , Shouxin Liua, Jian Lia, Mengyao Dongb,c, Jincheng Fanb,d, Zhanhu Guob a Key Laboratory of Bio-based Material Science and Technology of Ministry of Education, College of Material Science and Engineering, Northeast Forestry University, Harbin, 150040, China b Integrated Composites Laboratory (ICL), Department of Chemical and Bimolecular Engineering, University of Tennessee, Knoxville, TN, 37996, USA c Key Laboratory of Materials Processing and Mold (Zhengzhou University), Ministry of Education, National Engineering Research Center for Advanced Polymer Processing Technology, Zhengzhou University, Zhengzhou, 450001, China d College of Materials Science and Engineering, Changsha University of Science and Technology, Changsha, 410114, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Fungus fibers N,S-self-doped carbon dots Biosynthesis Tetracyclines Cellular imaging

In this work, nitrogen and sulfur dual-doped carbon quantum dots (N,S-CDs) from naturally renewable biomaterial fungus fibers were prepared by a biosynthesis and hydrothermal method. The N,S-CDs displayed good water solubility, excellent stability, high quantum yield (QY = 28.11%) as well as remarkable features for fluorescence quenching-based detection and cellular imaging of cancer cells. It was worth mentioning that the heteroatoms doped carbon quantum dots made from the fungus fibers had a satisfactory QY and could be used as a selective, efficient, and sensitive fluorescent probe to determine tetracyclines by the synergistic effects of static quenching and internal filtration effect. The probe demonstrated a wide linear range and low detection limit. For tetracycline, the linear range was 0.5 μM to 47.6 μM, and the corresponding detection limit was 15.6 nM. Significantly, the test papers prepared by using N,S-CDs could detect tetracyclines in aquiculture wastewater rapidly. The produced N,S-CDs did not affect the cell viability and showed great promises for cellular imaging.

1. Introduction With the increasing demand for aquatic products (such as fishes, shrimps, etc.), high-density farming with intensive culture and feeding has been developed, and antibiotics have been extensively used in aquaculture for the prevention and treatment of fish diseases or to promote fish growth and reproduction [1,2]. Tetracyclines (TCs) serve as broad-spectrum antibiotics that are antibacterial to both gram-positive and gram-negative organisms, and are one of the main antibiotics used in aquaculture because of their good oral absorption and relatively low toxicity and low cost [3]. TCs include tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), and doxycycline (DOXC), and the first three are most commonly used in aquaculture. Excessive use of TCs could lead to unsafe residue levels [4,5]. Furthermore, the residues can also promote the development of bacterial resistance to antibiotics [6]. Therefore, it is particularly important to detect the TCs residues before the discharge of aquaculture wastewater. To date, various methods including high-performance liquid

chromatography [7], liquid chromatography-tandem mass spectrometry [8], high-performance liquid chromatography coupled with a tandem mass spectrometer [9], ultrahigh performance liquid chromatography methods [10], chemiluminescence [11], assay-based aptasensor [12], and colorimetric analysis [13] have been developed for the specific and sensitive detection of TCs. Nevertheless, these techniques also have some shortcomings including sophisticated instrumentation, complicated sample preparation, expensive, or/and skilled personnel needed that limited their practical applications. Thus, it is highly indispensable to develop a convenient, reliable, time-saving, cost-effective, and alternative method to be applied as excellent selective and sensitive probes for detecting TCs in aquaculture wastewater. Carbon quantum dots (CDs) are a novel nanomaterial with strong and tunable luminescence properties that have attracted considerable attention because of their comprehensive applications in the fields of fluorescent sensing and biological imaging [14,15], catalysis [16,17], drug delivery [18,19], energy [20,21], adsorption [22], and optronics [23]. Due to excellent light stability, adjustable emission wavelength,

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Corresponding authors. E-mail address: [email protected] (Z. Huang). 1 Cai Shi and Houjuan Qi are co-first authors. https://doi.org/10.1016/j.msec.2019.110132 Received 28 January 2019; Received in revised form 5 August 2019; Accepted 23 August 2019 Available online 31 August 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.

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executed to research the optical performance of N,S-CDs. The details of the characterization were described in the Supporting Information.

good water-solubility, and low toxicity, CDs had been widely utilized as fluorescent nanoprobes to detect various compounds quantificationally which were previously detected by fluorescent dyes and semiconductor quantum dots [24]. Such applications in detection are based on the principle that the interaction between the surface functional groups of CDs and analytes results in the quenching or enhancement of the fluorescence of CDs. Heteroatom doping has testified to be an effective method to strengthen material properties (including electrochemical and adsorption properties, etc.) [25–31], so it is possible to treat the surface of the CDs with heteroatoms (such as nitrogen, phosphorus, sulfur, boron, and fluorine, etc.) to improve their fluorescence properties. In recent years, various heteroatoms have been reported to dope CDs [32–34], but most of them require the modification by foreign chemicals. Self-doping of nitrogen and sulfur appears to be the most attractive and promising. However, the use of natural biomass as carbon sources often fails to meet this requirement. Fungus fibers prepared by biosynthesis are a filamentous material that is rich in proteins, amino acids, polysaccharide, polysaccharides, and vitamins. Because of its constitutions and the fluffy filaments of its interior, fungus fibers can be easily carbonized into N,S-CDs with strong photoluminescent properties, which are also reported in a class of biofibers that underwent similar fluorescent quenching under different conditions [35–37]. It is well known that nanomaterials are particularly sensitive to the type of starting materials, CDs derived from different precursors have different structures and vary in the application fields [38]. The biomaterials (fungus fibers) could be obtained at any time and any place with a constant mass, ensuring the reproducible preparation of nanomaterials. Furthermore, the nutrients of fungus fibers were synthesized through a biosynthetic process without any additional chemical substances, this method was simple, inexpensive, environmentally friendly, and was suitable for high-volume production. In this study, a green and straightforward approach was reported for the synthesis of water-soluble N,S-CDs with high resistance to photobleaching, ion strength, metal ions, and biomolecules from fungus fibers synthesized through biosynthesis. The obtained N,S-CDs were utilized as a novel fluorescent probe for determining TCs in aquaculture wastewater on the basis of the interaction between the surface functional groups of N,S-CDs and TCs. The N,S-CDs also exhibited promising potentials for HepG2 cells multicolor imaging. Furthermore, the excellent water solubility of the N,S-CDs along with its high recovery in standard samples made it possible to quickly detect TCs in complex environments using simple fluorescence detection test strips.

2.4. Quantum yields measurements The quantum yield (QY) of N,S-CDs was determined on the basis of an established procedure. The measurement details were shown in the Supporting Information. 2.5. TCs detection The detection of TCs was performed at room temperature. The sensing solution was prepared by adding TCs solutions of different concentrations into N,S-CDs solutions. After mixing thoroughly (about 10 s later), the fluorescence spectra of the solutions containing N,S-CDs and TCs were measured at 360 nm. The selectivities for TCs were determined in a similar way by adding other antibiotics and foreign biomolecules instead of TCs. Some common metal ions and anions that co-existed in aquiculture wastewater were also used to detect the selectivity for TCs. To investigate the detection of TCs by the N,S-CDs sensors in real samples, we simulated aquaculture wastewater in the laboratory according to the literature [39], and prepared test papers using N,S-CDs solution to determine TCs rapidly. All details were described in the Supporting Information. 2.6. Cell viability assay and cellular imaging of the N,S-CDs The cytotoxicity of N,S-CDs on cells was estimated by an MTT (3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide). The HepG2 cells were cultured for cellular imaging. All of the details of the experiment were shown in the Supporting Information. 3. Results and discussion 3.1. Structural characterization of N,S-CDs As presented in Fig. 1a, the TEM picture shows that the N,S-CDs had a narrow size distribution and were nearly spherical. The observed carbon lattice fringe spacing distance of 0.21 nm using HRTEM (Fig. 1b) was in agreement with that of graphitic carbon, indicating the graphitic nature of the N,S-CDs. As displayed in Fig. 1c, the Fast Fourier Transform (FFT) pattern also confirmed that the N,S-CDs were single crystals, which were in good agreement with the results of HRTEM image [40–43]. The size distribution (inset of Fig. 1a) of N,S-CDs obtained by software (Zeta sizer software, Ver. 2.2 from Malvern) indicated that the N,S-CDs were within the scope of 5.5 to 7.5 nm, and the average diameter was 6.5 ± 0.5 nm, indicating that the N,S-CDs were nearly uniform in size. The chemical compositions of N,S-CDs were analyzed by FTIR and XPS. Fig. 2a shows the FTIR spectrum of the synthesized N,S-CDs. The broad band in the region of 3052–3624 cm−1 was ascribed to the OeH and NeH stretching vibrations. The existence of these functional groups on the N,S-CDs contributed to their good water solubility. The absorption bands at 2937 and 2863 cm−1 were assigned to the stretching vibration of CeH. The bands at 1665 and 1583 cm−1 were corresponding to the C]O and C]N stretching vibration modes, respectively. The peak centered at 1395 cm−1 was identified as the stretching vibration of CeN, which confirmed the presence of nitrogen doping on the N,S-CDs. A broad peak at approximately 1000–1126 cm−1 corresponded to the –SO3 or C]S groups, and the bands at 627 and 2144 cm−1 were assigned to the CeS and SeH stretching, which indicated the doping of sulfur [40,44,45]. To gain an insight into the information obtained by FTIR, XPS was performed to further identify the chemical bonds and elemental composition present in the N,S-CDs. The C1s(284.6 eV) was used as a

2. Materials and methods 2.1. Materials All reagents were analytical grade or better and used without further purification. The detailed information was described in the Supporting Information. 2.2. Synthesis of N,S-CDs N,S-CDs from fungus fibers were prepared by a biosynthesis and hydrothermal method. Firstly, fungus fibers were prepared according to biosynthetic pathway. Subsequently, the N,S-CDs were synthesized from fungus fibers by the hydrothermal treatment. The details of the experiment were provided in the Supporting Information. 2.3. Characterization The morphologies of N,S-CDs were characterized by transmission electron microscope (TEM) and high-resolution transmission electron microscope (HRTEM). Fourier-transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS) were used to investigate the chemical structures. UV–vis spectra and fluorescence spectra were 2

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Fig. 1. (a) TEM image and particle size distribution; (b) HRTEM image of N,S-CDs and (c) FFT pattern of N,S-CDs from HRTEM image.

[42,43,48]. In addition, as evidenced by the S2p high-resolution XPS spectrum (Fig. 2f), sulfur was successfully doped in the framework of the CDs, which was resolved into two bands at 168.2 and 169.3 eV, demonstrating that sulfur was present as C–SOx species [46]. Consistent with the FTIR results, the XPS analysis results confirmed the presence of various functional groups containing oxygen and nitrogen, such as hydroxyl (–OH), carbonyl (–COOH), and amine (–NH2) groups in the N,S-CDs, which equipped this fluorescent materials with excellent water solubility, and made it easier to be modified, further promoted their applications in many fields.

reference to calibrate all binding energy values. The successful incorporation of N and S into the CDs was confirmed by the XPS analysis. Four predominant peaks of C1s, N1s, O1s, and S2p at 285, 400, 531, and 168 eV, respectively, were observed (Fig. 2b). The corresponding content of each element was 46.98%, 9.77%, 35.97%, and 7.28%, respectively (inset of Fig. 2b). Significantly, the amount of N (9.77%) and S (7.28%) was comparable to other biomass-based N,S-CDs reported in the literature [41,44,46,47]. This further demonstrated the advantages of this precursor. Specifically, the C1s high-resolution XPS spectrum in Fig. 2c could be fitted with four peaks approximately at 284.6, 285.5, 286.2, and 287.4 eV, which were assigned to the C=C/C–C, CeN, C–O/ C–S, and C=O/C=S, respectively. The N1s spectrum (Fig. 2d) were subdivided into three peaks. The peaks at approximately 399.1 and 400.5 eV corresponded to the pyridinic type (C–N–C, C]N) and the pyrrolic type (C3eN) N atoms, and the binding peak at 401.3 eV suggested the presence of sp3 hybridized nitrogen HeN. These results verified that the synthesized CDs were nitrogen-doped. The high-resolution O1s spectrum (Fig. 2e) revealed that the presence of C]O, C–O–H, and O=C–O/S–O at 530.9, 531.7, and 532.6 eV, respectively

3.2. Optical properties The optical performance of the synthesized N,S-CDs was surveyed by UV–vis and fluorescence spectroscopy (Fig. 3). In the UV–vis spectrum (Fig. 3a), the N,S-CDs exhibited two typical UV–vis absorption bands at 264 nm and in the range from 320 to 372 nm, which was ascribed to the π–π* transition of C]N or C]C bonds and the n–π* transition of –COOH or –NH2 groups on the surface of N,S-CDs,

Fig. 2. (a) FTIR spectrum of N,S-CDs; (b) XPS spectrum of N,S-CDs, and high-resolution spectra of (c) C1s, (d) N1s, (e) O1s and (f) S2p of N,S-CDs. 3

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Fig. 3. (a) UV–vis absorption spectrum, inset photograph of N,S-CDs aqueous solution under irradiation of daylight (left) and UV light (right); (b) Fluorescence excitation (blue line), and emission spectra (red line); (c) Fluorescence spectra of N,S-CDs at different excitation wavelengths. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

respectively. However, the peak at 264 nm led to no observable fluorescence, and the peak in the range between 320 and 372 nm, which corresponded well to the excitation spectrum, led to a strong luminescence emission [42]. As shown in Fig. 3b, the maximum emission band was focused at 440 nm with an excitation of 360 nm. Furthermore, the diluted N,S-CDs solution was pale yellow under visible light but radiated a bright blue luminescence under ultraviolet light at 365 nm (inset of Fig. 3a). The QY of the N,S-CDs was estimated as 28.11%, which was significantly higher than those of recently reported CDs derived from biomass, as presented in Table S1. The doping of CDs with heteroatoms could substantially improve the QY, as reported in the literature [40,43,46,49]. For example, Anh et al. [43] revealed that the introduction of N provided more electron-enriched activities, which promoted the high yields of the radiative recombination and reduced the nonradiative recombination, contributing to the high QY. Wang et al. [40] found that the QY of certain CDs was more likely to be affected by surface traps than by band gap modifications of the carbon core, and the relatively high QY of N,S-CDs was due to the introduction of –C=S instead of –SH. In this work, the increase in QY was related to the coexistence of N and S on the surface of CDs, which reduced the recombination of non-radiative electron-holes, and was also related to the high content of amino acids and proteins in biosynthetic materials. Fig. 3c shows the fluorescence spectra of N,S-CDs. The N,S-CDs exhibited an excitation-dependent emission character, and the strongest emission wavelength (440 nm) was obtained at 360 nm. Although it is challenging to explain the exact mechanism of the excitation wavelength-dependent emission behavior, these behaviors were probably related to the varied fluorescence characteristics of different sizes of nanoparticles or various surface-state emissive traps of CDs [32,50]. The stability of CDs in various conditions is an important factor for their applications in diverse applications. The fluorescence properties of the N,S-CDs were conducted under diverse conditions. First, the N,SCDs solution was continuously irradiated with a beam of ultraviolet light at 365 nm and only a negligible decrease in the intensity of fluorescence was observed after several hours (Fig. S1a), indicating the excellent photostability of N,S-CDs. Then, the influences of ionic strength and pH value of the solution (Figs. S1b and c) were investigated. As exhibited in Fig. S1b, the intensity of N,S-CDs fluctuated slightly even at a high ionic strength of 1.0 mol L−1, which was advantageous for practical applications in the existence of various salt solutions. As shown in Fig. S1c, the intensity of the N,S-CDs would be affected by strong acidic and strong alkaline environments, the reason might be that too many H+ and OH– would affect the functional groups on the surface of the N,S-CDs, the fluorescence intensity of N,S-CDs was inhibited, however, the N,S-CDs showed stable fluorescence within the pH scope of 3–10. The photographs of N,S-CDs aqueous solutions with different pH values from 1 to 12 showed that the color of the solution

remained bright blue when the pH was between 3 and 10 (inset of Fig. S1c). On the basis of above results, the N,S-CDs exhibited excellent stability, which was essential for accurate detection of analytes in complex environments. 3.3. Fluorescence response of N,S-CDs to TCs To investigate the sensing mechanism of this fluorescent probe, TC was chosen as a representative. The fluorescence spectra of N,S-CDs in the absence and presence of TC were studied (Fig. 4a). The N,S-CDs emitted bright blue luminescence with 365 nm ultraviolet light irradiation and the fluorescence was quenched drastically in the presence of TC (inset of the Fig. 4a). In the fluorescence emission spectra (Fig. 4a), the quenching efficiency was approximately 86% by calculation upon the addition of 100 μM TC, further indicating that TC could significantly inhibit the fluorescence of CDs. The possible principle for the decreased intensity of N,S-CDs arose from the specific interaction between TC and the functional groups on the N,S-CDs, which resulted in a strong intermolecular force. Fig. 4b shows the UV–vis spectra of N,S-CDs, TC, their mixture and the sum of the first two spectrum. It could be seen that the absorption of N,S-CDs increased dramatically with the addition of 25 μM TC. The spectrum of the mixture was similar to the theoretical spectrum, but the absorbance of the mixture was lower than the theoretically calculated value, indicating a possible static quenching. In order to comprehend the quenching principle by TC deeply, the fluorescence emission intensity was conducted based on the Stern–Volmer equation: F0/ F=Ksv[Q] + 1, where F and F0 were the fluorescence intensity of N,SCDs at 440 nm in the presence and absence of TC, respectively, Ksv was the Stern–Volmer quenching constant and [Q] was the concentration of TC. As show in Fig. 4c, it was evident that the fluorescence intensity of N,S-CDs was reduced with the concentration of TC augmented. However, both static quenching and dynamic quenching could be fitted to Stern–Volmer curve, so it could not be determined only by equation. Furthermore, fluorescence lifetime experiments were performed to get an in-depth insight into the quenching principle. The average fluorescence lifetimes of the N,S-CDs was calculated as 6.314 and 7.034 ns in the absence and presence of TC, respectively. There was no distinct change after adding TC (Fig. 4d), indicating that there was a groundstate interaction between N,S-CDs and TC, that is, static quenching [51,52]. The fluorescence decay curve was fitted by a double-exponential function, as shown in Table S2. Based on the above ultraviolet and fluorescence analysis, it was found that the TC solution showed an absorption peak from 250 to 400 nm (Fig. 4b), and the fluorescence excitation spectrum of N,S-CDs displayed a peak from 310 to 420 nm (Fig. 3b). Evidently, the absorption band of TC partial overlapped with the fluorescence excitation spectrum of N,S-CDs. It has been demonstrated that the fluorescence 4

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Fig. 4. (a) Fluorescence spectra of the N,S-CDs and N,S-CDs + 100 μM TC. The inset photographs of the N,S-CDs in the absence (left) and presence (right) of 100 μM TC under UV light of 365 nm; (b) UV–vis spectra of N,S-CDs, TC, their mixture and the sum of the first two spectrum; (c) Stern–Volmer curves of N,S-CDs in the presence of TC; (d) Fluorescence lifetime experiments of N,S-CDs (black line) and N,S-CDs + TC (red line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

fluorescence by interacting with the functional groups of N,S-CDs. The impacts of various negative ions and common metal ions were also investigated. As shown in Fig. S3b, the negative ions and common metal ions had no obvious effect on the fluorescence of N,S-CDs. These consequences confirmed the excellent selectivity of N,S-CDs for the detection of TCs. Prior to testing the applicability of the developed method, the analytical performance of it was evaluated. Under the same conditions, the fluorescence emission spectra of N,S-CDs were recorded after adding of different concentrations of TCs. The fluorescence intensity of the N,S-CDs gradually decreased with the addition of TCs (Figs. 5a, S4a, and S4c). The relationship between the fluorescence recovery factor F/ F0 and the concentrations of TCs was presented in Figs. 5b, S4b, and S4d. There was a good linear relationship between F/F0 and the concentration of TCs within the scope of 0.5–47.6 μM with a good correlation coefficient (R2 = 0.998, 0.992, and 0.991 for TC, CTC, and OTC, respectively). The limit of detection (LOD) was 15.6, 36.4, and 40.7 nM based on LOD = 3σ/k, respectively, where σ was the standard deviation of bank signals of the N,S-CDs (n = 11) and k was the slope of the calibration curve. The comparison of the analytical performance of TCs determination with other methods is presented in Table S3. Although the detection limits of the TCs are not very satisfactory, this method has many irreplaceable advantages, such as a wider linear range, environmentally friendly preparation method and excellent stability. It should be specially stated that compared with the reported similar N,SCDs (using chemical substances such as C3N3S3 [53] and glutathione [54] as raw materials, respectively), the prepared N,S-CDs in this study not only have the advantages of green renewable of biomass raw materials, but also can be used for the detection of three antibiotics, including TC, CTC, and OTC, and their detect limits and detection ranges

quenching also comes from the absorption of fluorescence by the quencher, i.e. inner filter effect [50]. In summary, the fluorescence quenching of N,S-CDs caused by TC was the result of a combination of static quenching and internal filtration effects. 3.4. Fluorescence detection of TCs Before the detection of TCs, a series of experiments were performed to optimize the detection conditions. The pH value has an effect not only on the surface charge of the N,S-CDs but also on the interaction between N,S-CDs and the target species. Therefore, it was necessary to investigate the effect of pH on the TC detection. The experiments were performed at different pH with and without TC. As shown in Fig. S2, when the pH was within the scope of 3 to 10, the intensity of N,S-CDs was relatively stable, and the sensitivity of the probe for the detection of TC was generally consistent across the entire pH range, which was favorable for developing a stable sensing method. It could be seen that the pH range of the method was relatively wide and advantageous for practical applications. The selectivity of fluorescent probe was crucial for the measurement of TCs in aquaculture wastewater. Thus, the potentially co-existed interferences (e.g. erythrocin, chloramphenicol, streptomycin sulfate, ampicillin Na, gentamicin sulfate, TC, OTC, CTC, bovine serum albumin, ascorbic acid, cysteine, lysine, glycine, CO32−, SO42−, NO3−, PO43−, Cl−, HCO3−, HPO42−, Cu2+, Fe2+, Zn2+, and Mn2+) were selected to assess the selectivity of the method. As shown in Fig. S3a, TCs could significantly quench the fluorescence, whereas other antibiotics or foreign biomolecules had no or little quenching effect on the fluorescence intensity of N,S-CDs. Because all TCs have a similar chemical composition and spatial structure, all of them could quench the 5

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are also comparable. Based on the detection performance of N,S-CDs above, it is feasible to apply this probe for determine TCs in real samples.

3.5. Detection of TCs in real samples To demonstrate the reliability of our method, the detection of TCs in aquaculture wastewater was carried out. The aquaculture wastewater samples were processed according to Section 2.5. Different concentrations of TCs were spiked in the samples and then analyzed by the proposed method above. As summarized in Table 1, the recoveries of TC, CTC, and OTC were in the range of 96.9–105.7%, 90.5–110.5%, and 91.6–107.5%, respectively, with relative standard deviations (RSD) less than 3.1%, 3.2%, and 3.0% (n = 3), respectively. Also, test papers were successfully designed for rapid detection of TCs in aquaculture wastewater using filter papers. The test papers were immersed in aquaculture wastewater containing different concentrations of TC (from left to right 0, 0.1, 10, and 100 μM; Fig. 6). When there was no TC, the test paper displayed blue fluorescence under ultraviolet light radiation. As the concentration of TC increased, the color of the test papers was gradually darkened. These results indicated that the sensing system is a powerful and efficient approach to detect TCs in the environmental samples.

Fig. 6. The photograph of N,S-CDs-based test papers under a UV lamp.

3.6. Cell cytotoxicity and fluorescence imaging In order to explore the potential application of N,S-CDs in bioimaging, the cytotoxicity of N,S-CDs was researched by HepG2 cells using MTT assay. As presented in Fig. 7a, the cell viabilities of the HepG2 cells were examined after exposure to various concentrations of N,SCDs, the cell viabilities remained over 95% even at a N,S-CDs concentration of 400 μg mL−1 after incubation for 24 h. These observations clearly showed that the synthesized N,S-CDs had a low cytotoxic effect on the cells. What's more, the concentration of N,S-CDs required for the potential bioimaging application is much lower. These results manifested that the N,S-CDs might be promising candidates for bioimaging applications. Based on the good biocompatibility, excellent photostability, excitation-dependent fluorescence behavior, and small size distribution of N,S-CDs, multicolor cellular imaging was conducted to verify the potential of N,S-CDs in bioimaging. Fig. 7b displays the bright-field and fluorescence pictures of HepG2 incubated with N,S-CDs. As expected, the stained HepG2 exhibited blue, green, and red fluorescence under the excitation of 435, 525, and 597 nm light, respectively, which proved that the N,S-CDs were efficiently internalized by HepG2 cells, i.e., N,S-CDs entered the HepG2 cells. Nevertheless, the fluorescence was turned off upon exposure of HepG2 to 100 μM of TC, showing that TC could be detected inside the living cells (Fig. 7b). These results further confirmed the potential application of N,S-CDs in bioimaging.

Table 1 TCs detection in aquiculture wastewater. TCs concentration Sample TC

CTC

OTC

Added (μM)

Found (μM)

RSD (%, n = 3)

2 4 10 20 40 2 4 10 20 40 2 4 10 20 40

2.11 3.95 9.69 21.13 38.92 1.81 4.12 11.05 18.52 36.44 2.15 4.08 9.16 18.84 40.23

2.8 3.1 2.5 2.9 3.0 2.6 3.2 2.3 3.0 3.1 2.5 1.8 2.7 3.0 2.2

Recovery (%) 105.5 98.75 96.9 105.65 97.3 90.5 103 110.5 92.6 91.1 107.5 102 91.6 94.2 100.58

4. Conclusions N,S-CDs from fungus fibers were successfully prepared by a biosynthesis and hydrothermal approach for the first time. The synthesized N,S-CDs with a high QY possessed good biocompatibility, good water solubility, and outstanding photostability, and could be used for 6

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Fig. 7. (a) Cell viability of HepG2 cells after incubation with N,S-CDs at varying concentrations of 24 h using an MTT assay (mean ± SD, n = 3); (b) fluorescent images of HepG2 cells incubated with N,S-CDs at the excitation wavelengths of 435, 525, and 597 nm. Scale bar: 50 μm.

References

cellular imaging of cancer cells and for detecting intracellular TC without any further modification. Moreover, the N,S-CDs were employed for assaying TCs owing to the synergistic effects of static quenching and internal filtration effect. The sensing system showed excellent selectivity and sensitivity towards TCs. These results demonstrated that the N,S-CDs derived from fungus fibers could be used as an ecofriendly material in biological and environmental applications in the future.

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Acknowledgments This work was financially supported by the National Natural Science Foundation of China (No. 31670592), the Natural Science Funds for Distinguished Young Scholar of Heilongjiang Province (No. JQ2019C001), and the Central University Basic Scientific Research Business Expenses Special Funds Project of China (No. 2572017EB03). Declaration of competing interest None. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.msec.2019.110132. 7

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