3.2 Optimization of the reaction parameters to prepare CDs
CDs were prepared using the freeze-ground fish-scale powder as a precursor, and the conventional hydrothermal and microwave preparation methods were further optimized.
3.2.1 Optimization of reaction parameters for CD-HT
CD-HT was prepared by a carbonization reaction under hydrothermal conditions with fish scales as a precursor. The effects of hydrothermal temperature, hydrothermal time, and NaOH concentration on the quantum yield were studied (Figure S2). Based on univariate experiments, NaOH concentration (0.03 mol/L, 0.05 mol/L, 0.1 mol/L), hydrothermal temperature (180℃, 190℃, 200℃), and hydrothermal time (2 h, 3 h, 4 h) were selected and analyzed using an orthogonal table L9(34).
The orthogonal experiment was analyzed using range analysis and variance analysis (Table S1). Hydrothermal temperature was determined to be the main factor affecting the quantum yield of CDs, followed by NaOH concentration and hydrothermal time. Among the nine trials in the orthogonal test, the preferred combination was determined to be a NaOH concentration of 0.03 mol/L, reaction time of 4 h, and temperature of 200℃. The quantum yield of CD-HT at these conditions was 6.04 ± 0.05%.
3.2.2 Optimization of reaction parameters for CD-MW
Similar to the study performed for CD-HT, the effects of microwave conditions (NaOH concentration, microwave power, microwave time) on the quantum yield of CD-MW were analyzed using single-factor analysis, and the results were shown in Figure S3. NaOH concentration (0.01 mol/L, 0.03 mol/L, 0.05 mol/L), microwave power (240 W, 400 W, 560 W), and microwave time (1 min, 1.5 min, 2 min) were selected using the orthogonal table L9(34) for orthogonal experimental analysis.
Results of the orthogonal experiment (Table S2) suggested that NaOH concentration with the highest R-value had the greatest influence on the fluorescence quantum yield. The influence of the other factors (microwave time and microwave power) on fluorescence quantum yield was secondary. After the verification test, the optimal scheme was as follows: NaOH concentration, 0.01 mol/L; microwave time, 1.5 min; microwave power, 400 W. The quantum yield of CD-MW using these parameters was 5.10 ± 0.13%.
3.3 Optical properties of CDs
The optical properties of the two CD solutions were compared and the results are shown in Fig. 1. The UV-Vis absorption spectra reveal that both CDs have a weak wide absorption band at 250 ~ 290 nm, which may be attributed to the π-π* transition of the C = C bonds during aromatic sp2 hybridization.(Pandiyan et al. 2020, Reckmeier et al. 2016, Shamsipur et al. 2018) The fluorescence properties of the two CDs were further studied based on their fluorescence spectra. The optimal excitation of CD-HT was 360 nm and the optimal emission was 430 nm, whereas the optimal excitation of CD-MW was 310 nm and optimal emission was 400 nm. The difference was related to the particle size, domain size, and surface states of the CDs.(Ma et al. 2022, Pundi &Chang 2022) The illustrations are images of the two CD solutions visualized using a fluorescent lamp and an ultraviolet lamp. The two CD solutions appeared transparent in visible light and were evenly dispersed without any aggregation or precipitation. Furthermore, they exhibited a bright blue fluorescence under ultraviolet light (365 nm). The fluorescence intensity of CD-HT was stronger than that of CD-MW, which was consistent with their quantum yield.
The fluorescence characteristics of both CDs were analyzed (Fig. 2). When the excitation wavelength of CD-HT increased from 320 nm to 360 nm, the fluorescence intensity increased first and then decreased, and the emission peak redshift was observed. The emission spectra of CD-HT exhibited excitation-related properties, which may be due to the difference in the size of the CDs and the presence of different organic groups on the surface of the CDs that resulted in different surface state distributions.(Khan et al. 2019, Liu et al. 2011) Fluorescence spectrometry of CD-MW revealed that when the excitation wavelength was increased from 280 nm to 350 nm, the corresponding emission positions at different excitation wavelengths were almost unchanged, and a single fixed emission at 400 nm was observed, indicating an almost excitation-independent emission behavior. The nonexcited-state behavior may be because the luminescence properties of CD-MW depend on the surface state rather than the quantum size effect, likely because the amino group enhances the surface functionalization of the carbon surface group, and the surface state of these CD-MW should be fairly uniform.(Karami et al. 2020, Krysmann et al. 2012, Yang et al. 2020)
The stability of the fluorescence performance of CDs is crucial for practical application.(Li et al. 2019, Thulasi et al. 2020, Yun et al. 2022) Therefore, we investigated the stability of both types of CDs under light and ion interference (Fig. 3). The two CDs were continuously excited under a xenon lamp for 120 min to measure their photostability. The luminescence intensity was almost unchanged, indicating the photostability of the prepared CDs. Next, the effect of ionic intensity on the emission spectrum of CDs was determined, and the fluorescence intensities of both CD variants were monitored at different salt concentrations. When the concentration of NaCl was increased from 0.2 mol/L to 1.0 mol/L, the fluorescence intensity was almost unchanged. In addition, both CDs yielded uniformly clarified solutions without obvious precipitation under the interference of salt. This finding showed that both CD variants had stable fluorescence characteristics at higher salt concentrations. Collectively, our findings revealed that both CD variants exhibited light stability and salt tolerance, which are important characteristics for their practical application.
In summary, both CDs exhibited good light stability and salt tolerance, but their optical properties were different. The excitation was inconsistent (CD-HT excitation at 360 nm and CD-MW excitation at 310 nm); CD-HT exhibited emission behavior related to excitation, whereas CD-MW exhibited emission behavior that was almost independent of excitation. Moreover, the quantum yield of CD-HT was slightly higher than that of CD-MW. This may be closely related to the surface state and also likely affected by the size. Thus, in our subsequent analysis, we will focus on the structural differences between CDs.
3.4 Morphology of CD-HT and CD-MW
The crystal structure of the CDs was analyzed using XRD and the results are shown in Fig. 4. Both CDs showed wide and obvious diffraction peaks, and the diffraction angle was 2θ = 21.55°, which, corresponding to the layer spacing d was 0.41 nm. Compared with bulk graphite (about 0.34 nm), the spacing of fluorescent carbon nanoparticles is larger, which may be due to the abundance of O and N groups, resulting in the weak aromatic-layer interaction between the graphite layers.(Rodríguez Padrón et al. 2018, Yang et al. 2018) Therefore, both CDs have the characteristics of amorphous carbon, and the degree of graphitization is low.
The size and structure of CDs were analyzed using TEM (Fig. 5). It can be seen from the TEM diagram that CD-HT is mostly uniform and appears as quasi-spherical nanoparticles with good dispersion. The histogram of size distribution indicated that the particle size-distribution range of CD-HT was 1.7–6.5 nm, average size was 4.02 ± 0.89 nm, and the average diameter was < 5 nm. On the other hand, CD-MW was more inclined to be uneven and appeared as irregular sheets, with a particle size distribution range of 2.4–7.2 nm and an average particle size of 4.23 ± 1.03 nm. The significant differences in morphology between both CD variants may be the main source of their optical properties.
3.5 Surface properties of CD-HT and CD-MW
The chemical structure and state of the elements in the CDs were analyzed using XPS. The XPS full spectrum (Figure S4) shows that the prepared CDs contain C, N, O, and Na. A small amount of Na was derived from the residual Na ions during the preparation of CDs. Ca and P were not found in the prepared CDs, indicating no obvious entrainment of these elements into the complex amorphous carbon structure during CD synthesis. This could be attributed to the NaOH system that was used. Under alkaline conditions, the solubility of the main inorganic component, hydroxyapatite, in fish scales was low, resulting in only trace Ca and P ions in the reaction system, which cannot easily participate in CD formation. Elemental composition analysis (Table 1) revealed that the self-doped N element in both CDs was considerable. The passivation effect of the doped elements conferred excellent optoelectronic properties upon the CDs.(Manioudakis et al. 2019) Further improvement showed that there were fewer doped elements in CD-HT than in CD-MW, which was likely related to the rapid reaction using the microwave method.
Table 1
Surface-element composition of the two CDs
Name | CD-HT | CD-MW |
C1s | 65.8% | 59.4% |
N1s | 13.1% | 16.5% |
O1s | 21.1% | 24.1% |
The high-resolution spectra of C1s, N1s, and O1s bands were deconvoluted as shown in Fig. 6. C element exhibited four chemical states at different peak positions: C-C/C = C near 284.8 eV, C-N near 285.9 eV, C-O near 286.5 eV, and C = O near 287.6 eV.(Fang &Zheng 2021) The content of different chemical states calculated by peak area are as follows: for CD-HT, the C-C/C = C content was 59.8%, C-N content was 10.9%, C-O content was 6.5%, and C = O content was 22.9%. For CD-MW, the C-C/C = C content was 39.8%, C-N content was 24.2%, C-O content was 8.5%, and C = O content was 27.3%. CD-HT contained more C-C/C = C, whereas the other three chemical states (structural defects) were less; these findings indicated that the doping of heteroatoms in CD-HT was less, which was consistent with the elemental analysis results of the total spectrum.
In addition to C, the N1s spectrum was fitted into four main peaks (Fig. 6B), which were attributed to pyridine N (398.5 eV), amine N (399.7 eV), pyrrole N (400.2eV) and quaternary N (401.3 eV).(Liu et al. 2020, Pillar-Little &Kim 2017) By analyzing the ratio of the different types of N, we found that CD-HT had a higher proportion of pyrrole N, conferring upon its better charge mobility and donor-acceptor ability, which is beneficial in the improvement of its quantum yield. Moreover, a small amount of oxidized N is present in CD-HT. The O1s spectrum (Fig. 6C) can be deconvoluted into two peaks at 531.1 eV and 532.4 eV, which are attributed to the presence of C = O and C-OH/C-O-C, respectively.(Liu et al. 2020) Oxygen in CD-MW tends to exist in the form of C-OH/C-O-C, whereas oxygen in CD-HT exists in the form of C = O, which is also conducive to the improvement of quantum yield.
The functional groups of CDs were further analyzed using FTIR, and the main signal regions that were identified are shown in Fig. 7.(Dutta 2017) The total spectrum (Figure S4) and specific peak parameters are shown in the Supporting Material (Table S3 and Table S4).
In Fig. 7A, the peak at ~ 3560 cm− 1 represents the O-H stretching vibration of -COOH in CD-HT, which is significantly stronger than that in CD-MW, indicating that CD-HT contains more -COOH, which is consistent with the results from XPS. For the signal peaks representing the N-H stretching vibration at ~ 3450 cm− 1, and N-H and O-H stretching vibrations at ~ 3300 cm− 1, the peak area of CD-HT is smaller than that of CD-MW, which is related to the less doping of N and O elements in CD-HT. The wider peak shape of CD-MW was attributed to higher hydrogen bond association, indicating the hydrophilicity of CD-MW. The signal peaks of = C-H and Ar-H (3060 cm− 1) and of saturated C-H asymmetric vibration (2860 cm− 1, ~ 2960 cm− 1, and 2944 cm− 1) in CD-HT were significantly stronger than those of CD-MW, which also indicated the high carbonization degree of CD-HT.
As shown in Fig. 7B, the peak area representing the C = C stretching vibration (~ 1650 cm− 1) in CD-HT is significantly larger than that in CD-MW, which is consistent with previous XPS results. There is no significant difference between the signal of N-H bending vibration and C-N stretching vibration of the amide at ~ 1560 cm− 1. The signal at ~ 1520 cm− 1 attributed to the asymmetric stretching vibration of -NO2 in CD-HT is significantly higher than that in CD-MW, indicating higher -NO2 content. The peaks in the range of 1480 ~ 1350 cm− 1 are mainly attributed to C-H bending vibration in -CH3/-CH2-; the corresponding signal peaks in CD-HT are more obvious, indicating a higher number of C-H bonds, which are closely related to its higher degree of carbonization. Additionally, the signal peak (1420 cm− 1) of C-O stretching vibration and O-H bending vibration in CD-HT attributed to -COOH is stronger than that in CD-MW, which is consistent with the analysis results of -COOH (Fig. 7A).
The above analyses revealed that due to the different heating methods used, CDs prepared from fish scales using the conventional hydrothermal and microwave methods exhibited obvious differences in optical properties, structural morphology, and surface group composition (Fig. 8).
Microwave heating can rapidly and uniformly increase the temperature of the reaction system and promote the carbonization of organic matter (mainly collagen) in fish scales to form CDs. The result of the rapid reaction is that the abundant N (~ 17%) in the organic matter is carried and doped into the product CDs to a greater extent, resulting in CDs with higher N doping. On the other hand, the slow heating using the conventional hydrothermal method was helpful in obtaining a more balanced product with lower local energy. Thus, N doping in CD-HT was less, but a relatively large proportion in the form of pyrrole N was present and a certain amount of -NO2 was formed.
Microwave heating puts forth higher requirements for reaction vessels. The use of traditional stainless steel sealed reaction vessels is limited, whereas conventional PTFE or glass containers cannot provide effective sealing strength. Thus, the temperature of the reaction system is usually only slightly > 100℃. Therefore, only a part of the protein (mainly collagen) could dissolve to form the nanosheet-like fluorescent material. TGA analysis of the precipitate in the reaction system of the microwave method showed a large amount of organic matter (Figure S5). In contrast, the heating temperature of the hydrothermal reaction has no obvious restrictions and can be preset to temperatures > 100°C. Therefore, CDs with higher carbonization degree, smaller size, and higher C = O/COOH content were obtained. These structural features are the main reasons for the higher quantum yield and lower excitation wavelength of CD-HT.