A green and facile one-step synthesis of multi-functionalized fluorescent carbon dots (CDs) was performed by using five different edible mushrooms as the sole carbon source without any additives. The chemical composition and morphological characteristics of the obtained mushroom CDs (MCDs) were characterized by using UV-vis absorption spectra, fluorescence spectrophotometer, Fourier transform infrared spectroscopy (FT-IR), and transmission electron microscopy (TEM). The results demonstrate that all the MCDs are spherical nanoparticles with average diameters in the range of 1-3 nm and all emit bright blue fluorescence with differences in the intensity. The surfaces of the MCDs are rich in hydroxyl, carboxyl or amino groups, which are originated from the diversity of mushroom ingredient. The five MCDs also exhibit excellent photostability and water solubility. Additionally, we found that the MCDs without further modification can be applied for highly selective detection of Pd²⁺ ion by the fluorescence quenching effect with a detection limit of 22.31 μM. This work shows the great potential of mushrooms as excellent raw materials in the development of multifunctional fluorescence carbon materials.

Carbon dots (CDs) are quasi-spherical carbon nanomaterial with particle sizes less than 10 nm (1). Due to their fascinating features, unique fluorescence, good water solubility, high stability and low toxicity (2), CDs exhibit potential applications in a variety of research fields ranging from sensing (3), bioimaging (4,5), drug delivery (6, 7), bacteriostasis (8) to photocatalysis (9,10). During the past decade, a range of synthetic strategies to produce CDs were reported, including chemical or electrochemical oxidation, laser irradiation, pyrolysis and microwave or ultrasonic assisted synthesis (11,12) by using a variety of carbon sources or precursors. To further improve the quantum yields of CDs, surface passivation, modification or doping with other elements, such as nitrogen, sulfur and phosphorus were developed (13–15). Among these methods, rigorous reaction conditions or complex post-treatment procedure is always required. In this respect, a greener and simpler preparation approach is highly desired for the synthesis of CDs.

Natural biological materials possess a high content of proteins, carbohydrates and lipids, making them ideal biocompatible carbon sources for production of CDs. And great efforts have been devoted to preparation of CDs by utilizing different natural biomass, including vegetables (cabbage, carrot) (16,17), fruit peels (watermelon, orange) (18,19), fruit juice (banana, papaya) (20,21), food (white pepper, garlic) (22,23), flavonoid nutraceuticals (Indian curcumin (24), plant leaves (willow, bamboo, Ginkgo biloba, aloe) (25–28), and animal derivatives (milk) (29). It is demonstrated that CDs originated from different natural biomass tend to show distinct optical properties or sensing abilities. Therefore, evaluation the properties of CDs derived from different biomass is still an interesting field and a major research focus.

Mushrooms are a class of high nutrition edible macro fungus. They contain bioactive and therapeutic compounds, such as polysaccharides, proteins, lipids, amino acids, vitamins, and flavonoids (30), thus can afford abundant chemical constituents, including carbon, nitrogen, oxygen, and phosphorus for the preparation of CDs (31). In addition, the amine and carboxyl groups of the raw materials can further provide a large number of reactive sites for potential biosensing applications (32). Recently, researchers have made some attempts on applying edible fungus, such as shiitake mushroom and oyster mushroom, as green carbon source for the preparation of fluorescence carbon dots, which showed potential applications in the detection of harmful heavy metal ions or volatile organic compound (33,34). However, there are quite a lot of mushrooms with different nutrient contents and it is worth further exploring whether the differences in mushroom composition will lead to variation in the fluorescence property of resulted CDs.

In this paper, we present a greener and simpler synthesis of five kinds of blue-emitting CDs through one step hydrothermal method by using mushrooms as the only carbon source without any additives. The selected mushrooms are White Hypsizygus marmoreus, Pleurotus ostreatus, Agaricus bisporus, Lentinus edodes and Pleurotus eryngii and the corresponding CDs are marked as W-CDs, O-CDs, A-CDs, L-CDs, E-CDs. Properties of the five mushroom CDs (MCDs), such as morphology and size, surface functional groups and fluorescence properties were fully investigated and compared. It is revealed that five blue-emission MCDs with differences in the fluorescence intensity, surface functional groups and stability were obtained. The MCDs are all spherical nanoparticles with diameters in the range of 1–3 nm. All the MCDs possess abundant hydroxyl and carboxyl groups, which made them all exhibit excellent water solubility. However, for W-CDs, O-CDs, L-CDs and E-CDs, there also exist some amino groups. The differences in surface functional groups originate from the components variation of different mushrooms. The fluorescence properties of MCDs were further compared and demonstrated that all the MCDs showed excellent stability upon time, NaCl and pH. And A-CDs displayed stronger fluorescence intensity and better stability than other MCDs. Furthermore, A-CDs was used to detect metal ions, which showed highly selectivity and sensitivity towards Pd²⁺ through fluorescence quenching effect. This approach using edible mushrooms as the sole carbon source to fabricate surface functionalized CDs provides a new avenue to construct label-free CDs of hazardous metal ions.

Materials and Methods

The materials and methods are deposited in Supplementary material at the bottom of the article.

Results and Discussion

Optimizing the Synthesis Conditions of MCDs

MCDs were prepared by one-step hydrothermal method using five kinds of mushrooms as the sole carbon source, as shown in Scheme 1. The synthesis conditions including reaction time, temperature and solid-liquid ratio have tremendous impacts on the fluorescence properties of MCDs, which are firstly optimized. L. edodes was used as template to optimize the reaction conditions. As shown in Figure S1, the fluorescence intensity of CDs varies with reaction time, temperature and solid-liquid ratio. With increasing the reaction time from 4 h to 12 h, the fluorescence intensity of L-CDs enhances gradually and reaches the maximum when the reaction time is 10 h (Figure S1a). Figure S1b displays the effect of reaction temperature on the fluorescence intensity of L-CDs. As increasing the reaction temperature from 160 °C to 220 °C, the fluorescence intensity of L-CDs continues to increase and reaches the maximum when the temperature reaches 200 °C. The optimized solid-liquid ratio is further tested (Figure S1c). With the change of solid-liquid ratio from 1:15 to 1:40, the fluorescence intensity of L-CDs goes to the maximum at the solid-liquid ratio of 1:20, then gradually decreases with the addition of more solvent. Thus, the optimal conditions for MCDs preparation are set as follows: 10 h, 200 °C and solid-liquid ratio is 1:20.

SCHEME 1: Schematic representation of MCDs synthesis.

FIGURE S1: Effect of reaction conditions on the fluorescence intensity of CDs: (a) reaction time, (b) reaction temperature, and (c) the solid-liquid ratio.

Optical Properties of MCDs

The ultraviolet-visible (UV-vis) spectra (Figure S2) show that all the MCDs exhibit two typical absorption peaks at c.a. 280 nm and 320 nm, which are attributed to the π-π* transition of sp² conjugated structures and the n-π* transition of the appearance of amine/carbonyl groups on the MCDs surface (35,36). For W-CDs, O-CDs, L-CDs and E-CDs, a weak peak at c.a. 250 nm is also observed, which may be ascribed to the π-π* transition of C=O bonds (37). The fluorescence spectra were further investigated and displayed in Figure 1a–e. All the MCDs exhibit the similar excitation wavelength dependent property. By increasing the excitation wavelength from 320 nm to 420 nm, the emission peaks of W-CDs, O-CDs, A-CDs, L-CDs and E-CDs gradually red-shift from 400 to 500 nm. The fluorescence intensity of W-CDs, O-CDs, L-CDs and E-CDs decreases with the excitation wavelengths varying from 360 nm to 420 nm, while the fluorescence intensity of A-CDs decreases as the excitation wavelength changing from 340 nm to 420 nm. By comparison, the optimum fluorescence intensity of A-CDs is much stronger than that of W-CDs, O-CDs, L-CDs and E-CDs. Under 365 nm UV light, the aqueous solutions of W-CDs, O-CDs, A-CDs, L-CDs and E-CDs all show blue light with slight difference in the intensity (Figure 1f). The quantum yields of W-CDs, O-CDs, A-CDs, L-CDs and E-CDs were 4.81%, 4.51%, 8.67%, 5.70% and 5.57%, respectively. It demonstrates that all the MCDs exhibit the similar fluorescence properties with slight differences in the intensity and emission peaks. As previously reported (38,39), the basic ingredients of different edible fungus are protein, amino acids and carbohydrates with differences in content, which can lead to the similar luminescence with differences in the intensity and emission peaks of various MCDs.

FIGURE S2: UV-vis spectra of MCDs.

Characterization of MCDs

FT-IR was used to characterize the surface groups of the obtained MCDs. As shown in Figure 2a and Table SI, the FT-IR spectrum of W-CDs shows characteristic stretching vibration of O-H, N-H, C-O at 3397 cm^-1, 3173 cm^-1, 1080 cm^-1, and the bending vibration of N-H, O-H at 1605 cm^-1, 1401 cm^-1 (40), respectively. For O-CDs, the peaks at 3420 cm^-1 and 3188 cm^-1 are ascribed to O-H and N-H stretching vibration respectively, and the peaks at 1632 cm^-1, 1401 cm^-1 and 1126 cm^-1 are attributed to the N-H, O-H bending vibration and C-O-C stretching vibration (41) (Figure 2b and Table SII), respectively. For A-CDs, the bands at 3439 cm^-1, 1640 cm^-1, and 1402 cm^-1 are attributed to the O-H, C=O stretching vibration and O-H bending vibration, respectively (Figure 2c and Table SIII). For L-CDs, the bands at 3416 cm^-1 and 3188 cm^-1 are assigned to the stretching vibration of O-H and N-H (42). The stretching vibration of C=O and the bending vibration of O-H are observed at 1659 cm^-1 and 1401 cm^-1 (Figure 2d and Table SIV). The FT-IR spectrum of E-CDs indicates that the band at 3420 cm^-1 is O-H stretching vibration, and the band at 3188 cm^-1, 1632 cm^-1 are ascribed to N-H stretching vibration and bending vibration (43). The O-H bending vibration and C-O-C stretching vibration are observed at 1401 cm^-1 and 1126 cm^-1 respectively (44) (Figure 2e and Table SV). The FT-IR results demonstrate the presence of hydroxyl, amine, carboxyl groups on the surface of W-CDs, O-CDs, L-CDs and E-CDs, and hydroxyl, carboxyl groups on the surface of A-CDs. The presence of these functional groups confers the hydrophilicity and aqueous solubility of MCDs (45). The differences in the surface functional groups of MCDs will lead to the variation of fluorescence property.

FIGURE 2: FT-IR spectra of (a) W-CDs, (b) O-CDs, (c) A-CDs, (d) L-CDs, and (e) E-CDs.

TEM was used to assess the morphology and size distribution of the as-prepared MCDs (3). TEM images of W-CDs, O-CDs, A-CDs, L-CDs, E-CDs revealed that they are spherical and homogeneously distributed particles with diameters in the range of 1–3 nm, and the computed average particle size are about 2.78 nm, 2.45 nm, 1.46 nm, 2.31 nm and 2.80 nm respectively. Furthermore, the HR-TEM images in Figure 3 (the inset) indicate that most of the MCDs have lattice fringes and in good crystalline state, while the lattice fringes of W-CDs are not found, demonstrating that the W-CDs nanodots are amorphous carbon (32). The lattice spacing of O-CDs, A-CDs, L-CDs, E-CDs are 0.18 nm, 0.21 nm and 0.23 nm, 0.20 nm and 0.20 nm respectively. For O-CDs, the lattice spacing of about 0.18 nm is close to that of (102) graphite carbon (46). A-CDs displays a lattice spacing of about 0.21 nm and 0.23 nm (Figure 3c inset), which are accompanied with the graphitic carbon (100) surface (47,48). The lattice spacing of L-CDs and E-CDs are 0.20 nm, which are identified as the (102) facet of graphitic carbon (49).

FIGURE 3: TEM images and the diameter distribution of (a) W-CDs, (b) O-CDs, (c) A-CDs, (d) L-CDs, (e) E-CDs, Inset for TEM images: high resolution-TEM images of MCDs.

Stability of CDs

In order to determine the stability of CDs in the environment, further experiments were carried out. The results showed that the fluorescence intensities of all the MCDs almost unchanged with different storage time (1 to 7 d), different concentration of NaCl (0.2-2.0 M) and pH value (2.2-10.6) (Figure 4a-c), indicating that all the MCDs had good stability and can provide a wide application prospect. Besides, the zeta potential of W-CDs, O-CDs, A-CDs, L-CDs and E-CDs are -16.5 mV, -11.2 mV, -33.7 mV, -12.1 mV, -13.0 mV respectively (Figure 4d). According to the FT-IR results, A-CDs possess hydroxyl and carboxyl groups, while the surface functional groups of other MCDs are amino, hydroxyl, and carboxyl groups. The differences of surface functional groups will lead to ζ-values change and the presence of amino groups can increase the electropositivity of MCDs. Thus A-CDs has the highest absolute ζ-value of 33.7 mV (50), and shows better stability that other four MCDs.

FIGURE 4: Effect of (a) different time, (b) concentration of NaCl, and (c) pH on the fluorescence intensity of MCDs; (d) Zeta potential of MCDs under the same conditions.

Metal Ion Sensing

Palladium as a transition metal has been widely used in the electrical and electronic industries, fuel cells and catalytic converters, which frequently release large amounts of palladium into the environment and can cause serious health problems (51). Therefore, it is of great significance to develop a simple and effective method for the detection of palladium. In this paper, five edible mushrooms as excellent biomass raw materials were used to prepare CDs, which all showed good fluorescence properties and stability. By comparing, A-CDs exhibits much stronger fluorescence intensity and stability, thus it is selected as a model to investigate the metal ion recognition property. When different metal ions with the same concentration (400 µM) were added into the A-CDs solution, the results were collected and shown in Figure 5. Pd²⁺ can effectively quench the fluorescence of A-CDs, while other cations have little effect on the fluorescence intensity of A-CDs. Furthermore, the monitoring of Pd²⁺ is not interfered by other metal ions (Figure 6). As illustrated in Figure 7a, with increase the concentration of Pd²⁺ ion from 0 to 400 μM, the fluorescence intensity of A-CDs gradually decreases. And a good linear range between F₀/F and Pd²⁺ concentration was obtained from 20 µM to 400 µM with the low detection limit of 22.31 μM (Figure 7b). This green-synthesized CDs with edible mushroom as the sole raw material without further modification provides a new and safer material for the detection of Pd²⁺.

FIGURE 5: Effect of different metal ions on the fluorescence intensity of (a) A-CDs and (b) the photographs of A-CDs with different metal ions under 365 nm UV lamp.

FIGURE 6: Selectivity of A-CDs towards Pd²⁺ over other metal ions.

FIGURE 7: Fluorescence response of (a) A-CDs in the presence of various concentrations of Pd²⁺ (0-400 µM), and (b) the relationship between F₀/F ratios versus Pd²⁺ concentration.

Conclusion

In this work, we successfully prepared MCDs from a variety of edible mushrooms by one-step hydrothermal method. Their properties were thoroughly investigated and compared. It is revealed that the mushrooms with differences in components will affect the properties of the prepared CDs, such as the presence of surface functional groups, the fluorescence properties and the stability. The green-synthesized A-CDs with Agaricus bisporus as the sole raw material without further modification shows highly sensitivity and selectivity towards Pd²⁺. This work reveals the great potential of mushroom as excellent raw materials in the development of multifunctional fluorescence carbon materials.

Acknowledgments

The authors would like to thank the Shandong Province Modern Agricultural Industry Technology System (SDAIT-07-07), Natural Science Foundation of Shandong Province (CN) (ZR2018LC022), Shandong Province Higher Educational Science and Technology Program (J18KA133), High-level Science Foundation of Qingdao Agricultural University (6631405), and National Natural Science Foundation of China (21403122, 32072292 and 31501331) for supporting this research.

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Supplementary Material for One-step Green Synthesis of Functionalized Carbon Dots from Five Edible Mushrooms

Materials and Methods

1. Chemicals

In this work, edible fungus (White H. marmoreus, P. ostreatus, A. bisporus, L. edodes, P. eryngii) are purchased from the local supermarket (Qingdao, China). Metal ions Cd(ClO₄)₂·6H₂O, Co(ClO₄)₂·6H₂O, Cu(ClO₄)₂·6H₂O, EuCl₃·6H₂O, Mg(ClO₄)₂, Mn(ClO₄)₂·6H₂O, Ni(ClO₄)₂·6H₂O, Zn(ClO₄)₂·6H₂O, and Pd(O₂CCH₃)₂ are obtained from Energy Chemical Technology Co. Ltd. (Shanghai, China). HgCl₂ is ordered from Aladdin Reagent Co. Ltd. (Shanghai, China). All other reagents in the experiment are analytical grade.

2. Optimization of Preparation Conditions

The preparation conditions, such as reaction time, temperature and solid-liquid ratio have significant effect on the properties of MCDs. Therefore, according to the reported preparation conditions (1,2), we selected L. edodes as the template to optimize the preparation conditions of L-CDs, including reaction time (4 h, 6 h, 8 h, 10 h, 12 h), temperature (160 °C , 180 °C, 200 °C, 220 °C) and solid-liquid ratio (1:15, 1:20, 1:25, 1:30, 1:40). Finally, the optimized preparation conditions of L-CDs were selected according to the fluorescence property of L-CDs.

3. Preparation of MCDs

All the mushrooms were pre-treated in a similar way. Firstly, edible mushrooms were cleaned with distilled water to remove impurities and cut into small pieces. After freeze drying, the mushrooms were ground into powder to prepare MCDs by a hydrothermal method. 0.25 g of dried mushroom powder was dissolved in 5 mL ultrapure water and then the mixture was added into Teflon-lined autoclave followed by heating at 200 °C for 10 h. After chill-down naturally to room temperature, the obtained brown solution was filtered to remove black residues and orange-yellow liquid was collected. Subsequently, the product was dialyzed against ultrapure water for 48 h. After, the dialysis solution was freeze dried in vacuum. Finally the CDs were dispersed in ultrapure water and store at 4 °C for further use.

4. Characterization

The morphology and size distribution of MCDs were observed with a transmission electron microscopy (TEM, Hitachi TEM-2100Plus, Japan) at 200 kV. The surface functional groups of MCDs were determined by Fourier transform infrared (FT-IR) spectrum on a Nicolet iS10 spectrometer (Thermo Fisher, USA) by potassium bromide pellet. The ultraviolet and visible spectrophotometry (UV-vis) spectrum was recorded on a U-3900 spectrometer (Hitachi, Japan). The fluorescence properties of MCDs were measured by F-2700 fluorescence spectrophotometer (Hitachi, Japan) with the slit widths of 10 nm. The stability of MCDs and recognition of metal ions were measured under the optimal excitation wavelength. Zeta potential of MCDs was measured using a Nano-ZS90 ZETASIZER (Malvern, UK). Absolute fluorescence quantum yields were performed on a FLS1000 fluorescence spectrometer (Edinburgh Instruments, England).

5. Metal ion sensing

Multiple metal ions (Pd²⁺, Co²⁺, Cu²⁺, Ni²⁺, Cd²⁺ , Eu³⁺, Hg²⁺, Mg²⁺, Zn²⁺and Mn²⁺) were used in the experiments. Metal ion solutions (2000 µM) were freshly prepared and 1.0 mL of metal ion solution was mixed with carbon dots solution (4 mL, 0.5 mg/mL). The mixtures were incubated at ambient temperature for 5 min. Then the fluorescence spectra were recorded from 350 nm to 700 nm at the excitation wavelength of 340 nm. In addition, the limit of detection (LOD) is calculated by 3σ/K, where σ is the standard deviation of the aqueous solution of A-CDs (n = 10), and K stands for the slope of the standard curve (3).

References

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2. Yang, K.; Liu, M.; Wang, Y.; Wang, S.; Miao, H.; Yang, L.; Yang, X. Carbon Dots Derived from Fungus for Sensing Hyaluronic Acid and Hyaluronidase. Sens. Actuators, B 2017, 251, 503–508. DOI: 10.1016/j.snb.2017.05.086
3. Qu, F.; Huang, W.; You, J. A Fluorescent Sensor for Detecting Dopamine and Tyrosinase Activity by Dual-Emission Carbon Dots and Gold Nanoparticles. Colloids Surf., B 2018, 162, 212–219. DOI: 10.1016/j.colsurfb.2017.11.055

Yingdi Gao, Fansheng Cheng, and Wenxiang Li are with the College of Food Science and Engineering at Qingdao Agricultural University, in Qingdao, China. Dan Zhu is with the College of Life Science at Qingdao Agricultural University, in Qingdao, China. Weina Li is with the College of Chemistry and Pharmaceutical Sciences at Qingdao Agricultural University, in Qingdao, China. Direct correspondence to: wnli@qau.edu.cn ●