English

• 0.   Introduction

  Carbon dots (CDs) are zero-dimensional carbon nanomaterials composed primarily of carbon, with nitrogen, sulfur, oxygen, and other elements as dopants. They are commonly referred to as fluorescent CDs due to their high biocompatibility, low toxicity, chemical stability, wide band absorption range, adjustable emission wavelength, and excellent fluorescence performance^[1-2]. CDs are nanocrystals smaller than 20 nm and consist of a graphitized sp² carbon core surrounded by a shell containing numerous functional groups such as hydroxyl, amino, carbonyl, and ether groups on the surface^[3-4]. These features make them easy to prepare and offer advantages such as optical properties that remain stable without bleaching^[5]. In contrast to many semiconductor quantum dots which may have certain toxicities due to their size or heavy metal content hindering targeted modifications^[6], CDs themselves do not contain toxic substances and can be sourced from a wide range of materials making them environmentally friendly new materials for various applications^[7-23]. With their exceptional electronic, optical, and physicochemical properties, CDs can also be modified with different functional groups on their surface for specific functionalization purposes, making them highly competitive research subjects at present. CD materials have been applied in various biological models, including cells, E. coli, mice, and zebrafish, making them good model organisms for studying biological imaging and fluorescent probes^[22-27]. Zebrafish, in particular, is an aquatic model organism developed in recent years known for its low environmental requirements, strong resistance to water temperature changes, and genetic conservation rate of 87%, earning it the nickname "mouse in water". Due to their high similarity to humans in terms of pharmacological and physiological responses, zebrafish possess significant persuasive power in toxicology studies. In comparison to traditional animal models such as rats and mice, zebrafish offer numerous advantages including the ability for in vitro fertilization and development, transparency, and a high reproduction rate (mature females can produce 100-300 eggs each time), ensuring an ample sample size for statistical analysis. Moreover, zebrafish are easier to care for and feed, making them widely utilized in embryonic toxicology research^[28].

  Kelp is renowned for its elevated protein content, balanced amino acid ratio, as well as its abundance of vitamins, minerals, and carotenoids that enhance the nutritional value of conventional food sources while influencing human and animal growth and health. Particularly within aquaculture settings, kelp serves as a common aquatic feed additive due to its rapid reproduction rate and exceptional nutritional composition. However, under normal circumstances, the large size of kelp poses challenges in effective dispersion or dissolution thereby limiting its full potential impact. Therefore by transforming kelp into fluorescent CDs, these nanoscale materials not only retain their advantageous characteristics such as similar size distribution with green emission properties but also inherit certain attributes from kelp itself. This unique combination presents promising prospects within the fields of aquaculture and biomedical science. There is growing interest among researchers regarding the potential toxicity risks associated with CD applications at biological levels^[29-31]. To further investigate the ecological toxicity of CDs on aquatic organisms specifically algae commonly found in aquaculture settings were selected as carbon and nitrogen sources during hydrothermal synthesis resulting in biomass fluorescent CDs. To investigate the fluorescence performance of CDs in zebrafish, we investigated their impact on morphological indicators of zebrafish embryo development, including hatching rate and heart rate aiming to determine the potential toxicity of CDs affecting zebrafish embryo development and provide insights for future biological and medical applications.

  1.   Methods

  1.1   Synthesis of CDs

  As shown in Fig. 1, kelp dry products were soaked in water for 24 hours before being gently dried with a paper towel to remove excess moisture. A total of 10 g kelp was homogenized with 50 mL pure water and transferred into a polyethene-lined stainless steel high-pressure reaction kettle, heated at 200 ℃ for 8 h. After cooling naturally to room temperature, the resulting sample was filtered using a membrane filter (0.22 μm) to remove impurities. The purified CDs solution was further dried at 60 ℃ using an oven-water bath system before being lyophilized to obtain brown powder samples.

  Figure 1

  

  Figure 1.  Schematic illustration of the synthesis route of CDs

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  1.2   Characterizations

  Transmission electron microscope (TEM) images of CDs were captured using a FEI-Talos F200s (Thermo Fisher Scientific) operated at 200 kV. With a D/Max-Ultima+ (Rigaku) diffractometer, an X-ray diffraction (XRD) pattern was obtained for CDs with a Cu Kα radiation (λ=0.154 056 nm) at a scanning rate of 10 (°)·min^-1 from 5° to 90°, operating at 40 kV and 40 mA. X-ray photoelectron spectroscopy (XPS) experiments were conducted using an Escalab 250Xi spectrometer (Thermo Fisher Scientific). Ultraviolet-visible-near infrared (UV-Vis-NIR) absorption spectra were measured using a UV-Vis spectrometer UV3600 (Shimadzu). Fluorescent spectra were recorded using a Fluoro Max-4 (HORIBA Scientific) luminescence spectrometer. Fourier transform infrared (FTIR) measurements were performed using a Nicolet 6700 (Thermo Fisher Scientific) spectrometer. A fluorescence microscope was used for the imaging experiments (LEICA DM4 B).

  1.3   Zebrafish breeding

  The wild AB zebrafish, purchased from Shanghai Fisher Bio Company, were cultured in a laboratory artificial climate box with a controlled temperature of (28±0.5) ℃ and a light cycle of 14 h (bright) / 10 h (dark). They were fed newly hatched shelled shrimp eggs six times a day. Suction was performed 0.5 h after each feeding to remove shrimp egg residue and zebrafish excreta using a large straw. Daily water changes were done by replacing one-third of the original amount. For the breeding and incubation of zebrafish, one female and more male adult zebrafish were put into the culture tank in the evening, and the middle of the tank was divided into two spaces, under the trough to prevent the barrier (prevent zebrafish from swallowing their eggs), placed the male and female zebrafish in two spaces in the tank respectively, and fed the newly hatched shrimp eggs later. On the second day at 8∶00 a.m., the partition was removed for mating between male and female zebrafish which lasted for approximately 1 h. After mating, collected eggs were transferred using a pipette gun to an artificial climate box where they underwent incubation at a controlled temperature of (28±0.5) ℃ until completion within 72 h.

  1.4   CDs exposure experiment of zebrafish

  For fluorescence imaging experiments involving CDs of zebrafish embryos, place normally developing zebrafish embryos that had reached the 4 h post-fertilization stage into individual wells, with 10 embryos per well in parallel for a total of 5 concentration gradients (0, 0.5, 1, 2, and 4 mg·mL^-1), each concentration gradient had 3 parallels, the exposure period lasted for 4 d, during which the exposure solution needed to be replaced every day. For experiments involving zebrafish embryonic metabolism, zebrafish exposed for 2 d were transferred to clear water and cultured until 4 d. After the experiment, a fluorescence microscope was used to observe the white field image and fluorescence field in CD-exposed zebrafish embryos. Took 60 adult zebrafish and divided them into two groups, with 3 parallel groups in each group. They were soaked in CD solutions of different concentrations (0, 1 mg·mL^-1) for 12 h, and the survival rates of adult zebrafish were calculated.

  2.   Results and discussion

  2.1   Structural characterization of CDs

  The morphology and size of CDs were observed by TEM. From Fig. 2a, the prepared CDs were within 3 nm and had the same size, showing excellent monodispersity and relatively uniform sphere shape. Fig. 2b shows the CDs′ XRD pattern, with a wide peak at 20°-30°, indicating that the CDs are amorphous carbon. XPS analysis further confirmed the composition of CDs, which consists of three elements: C, N, and O. The core levels corresponding to C1s (288.2 eV), N1s (402.6 eV), and O1s (534.4 eV) were identified (Fig. 3a). As shown in Fig. 3b, the C1s peaks of CDs can be divided into three single peaks, C—C (283.81 eV), C—N (sp²) (285.06 eV) and C=C (287.32 eV). In addition, the peak of N1s (Fig. 3c) can be divided into two single peaks, C—N (sp²) (398.92 eV) and N—H (400.82 eV). As for the peak of O1s (Fig. 3d), it can be divided into C=O (530.40 eV) and C—O (531.60 eV). It can be seen that CDs mainly contain C, N, and O, among which C is the most abundant element. XPS analysis shows that CDs consist of C, N, and O.

  Figure 2

  

  Figure 2.  (a) TEM image (Inset: the corresponding particle size distribution map) and (b) XRD pattern of CDs

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  Figure 3

  

  Figure 3.  (a) XPS survey spectrum, high‐resolution XPS spectra of (b) C1s, (c) N1s, and (d) O1s of CDs

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  The optical properties of CDs were characterized by UV-Vis absorption spectrum as well as fluorescence emission spectra at different excitation waves. As shown in Fig. 4a, CDs had distinct broad-band absorption peaks. As shown in Fig. 4b, with excitation light at wavelengths 350, 360, 370, 380, 400, and 420 nm, the emission spectrum of CDs is found to have excitation dependence, and the emission peak position also redshifts with the excitation wavelength with increasing excitation wavelength. The fluorescence emission spectrum showed that the maximum emission peak of CDs was 421.8 nm, so the fluorescence emission spectrum of CDs is consistent with the blue fluorescence results of CDs under ultraviolet light excitation. The results of FTIR spectroscopy testing of CDs (Fig. 4c) indicate the peak at 1 649 cm^-1 corresponds to the C=C in CDs, while the peak at 3 345 cm^-1 indicates the stretching vibration of —OH groups. Additionally, the peaks observed between 1 000-1 400 cm^-1 suggest the expansion of the —C—OH structure and the bending vibration of the —OH, indicating a large surplus of hydroxyl groups. The peak is consistent with the stretching vibration of —C—O—C— at 1 550 cm^-1, indicating that the CDs are partially oxidized on the surface during the experiment.

  Figure 4

  

  Figure 4.  (a) UV‐Vis absorption spectrum, (b) fluorescence spectra, and (c) FTIR spectrum of CDs

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  2.2   Fluorescence imaging of CDs in zebrafish

  CDs have excellent photophysical and photochemical properties, allowing them to be used for in vivo bioluminescent imaging. The application of CDs in vivo fluorescence imaging, influenced by concentration and time, has not been reliably studied in detail so far. To disentangle the imaging performance of CDs as bioluminescent markers, zebrafish was selected as a biological model in this experiment to study the fluorescence imaging effect of transparent embryos and luminescence of CDs in live zebrafish and the effect of different concentrations on the fluorescence imaging effect.

  The study of the fluorescence bio-imaging in different time points confirmed the spatial-temporal distribution of CDs, zebrafish embryos were exposed to different concentrations of CDs solution (0.5, 1, 2, and 4 mg·mL^-1) to observe the effect of CDs on zebrafish embryo development. As shown in Fig. 5, zebrafish embryos exposed to CDs showed stronger fluorescence compared with the control group (0 mg·mL^-1), while the fluorescence of the control embryos was significantly weaker than the CDs solution, indicating that CDs could enter the ooplasm of zebrafish embryos and had good biocompatibility. The fluorescence intensity was weak at 1 h, indicating that it takes some time for CDs to enter the embryo through the villous shell on the embryo surface. Along with the enhancement of the fluorescence intensity ascended. It was also found that no developmental damage was observed in zebrafish embryos exposed to CDs solution, indicating the good biocompatibility and biosafety of CDs.

  Figure 5

  

  Figure 5.  Long fluorescent imaging of zebrafish: (a, e, i) bright field and (b‐d, f‐h, j‐l) fluorescent field (scale bar: 250 μm)

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  The embryos had developed normally into small fish and still had a relatively strong fluorescence intensity at 48 h. Therefore, during this period, the 48 h exposed embryos were replaced with normal aquaculture water and continued to observe the metabolism of CDs in zebrafish. The fluorescence effect changed by the third day. The fluorescence intensity in the zebrafish gradually weakened and most of the fluorescence was only concentrated in the abdomen of the fish. As the time increased, the small fish continued to grow and develop, and only extremely weak fluorescence was observed in the abdomen. Until the fourth day, the small fish had only its fluorescence, the fluorescence intensity of the CDs disappeared almost completely (Fig. 6). Therefore, it is speculated that CDs may be recycled by the zebrafish digestive system, metabolic substances in its body, or other substances quencher. Fluoograms of CDs in zebrafish, showing some time requirement for its application to bioluminescent imaging. Through the fluorescence observation, it is seen that the metabolic process of CDs in small zebrafish is extremely slow, and the zebrafish embryos develop into small fish without any damage. In conclusion, we confirm that CDs are good biofriendly types that can be used as bioluminescent imaging for long observation material.

  Figure 6

  

  Figure 6.  Metabolism of CDs in zebrafish: (a) bright field and (b‐d) fluorescent field (scale bar: 1 000 μm)

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  2.3   Toxicology experiments of fluorescent CDs in zebrafish

  To analyze the data related to zebrafish, using SPSS 22.0 and performing a one-way ANOVA with a Duncan test. It can be seen from the above experiments that CDs can enter the zebrafish fertilized egg, which may affect the metabolic activity of life. For this purpose, 0, 0.5 mg·mL^-1 of CDs solution was experimentally selected to study the hatching rate of zebrafish fertilized eggs, using a six-well plate to culture 10 fertilized eggs per well for continuous observation and statistical hatching rate. Fig. 7a shows the hatching rate of zebrafish fertilized eggs in CDs solution. It showed that even after the zebrafish fertilized eggs were immersed in CDs solution, there was no obvious difference between the experimental group and the control group, indicating that CDs solution do not affect the hatching rate of zebrafish fertilized eggs; both the control group and the experimental group exhibited the phenomenon of death because under normal breeding conditions, there will be a small number of fertilized eggs that can not develop normally, which is a normal phenomenon. Therefore, the experimental results based on the survival rate show that CDs have excellent biocompatibility and have no adverse effects on the survival rate of zebrafish. By studying the effect of CDs on the heart rate of zebrafish, the heart rate of zebrafish within 15 s was selected for recording (Fig. 7b). The results showed that CDs solution had little effect on zebrafish heart rate and belonged to the normal fluctuation range. Fig. 7c shows that CDs solution (1 mg·mL^-1) exposure had no significant effect on survival in adult zebrafish compared with the control group and adult zebrafish showed better tolerance when coping with CD immersion. To sum up, CDs can enter zebrafish and zebrafish embryos, and the absence of observed embryo malformation indicates that the zebrafish embryos in 0.5 mg·mL^-1 are in a relatively healthy state of survival. This suggests that the experimental preparation of CDs exhibits good biocompatibility and biological safety, providing a reference for subsequent biological and medical applications.

  Figure 7

  

  Figure 7.  (a) Hatching rate, (b) heart rate of zebrafish embryos, (c) survival rate of adult zebrafish in CDs

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  3.   Conclusions

  Zebrafish live fluorescence imaging was used to test the effect of CDs in live zebrafish, and the location of CDs in zebrafish was analyzed. In vivo, fluorescence imaging results in zebrafish showed that our prepared CDs had good fluorescence imaging. The feasibility of CDs as bioluminescent probes and the stability of live fluorescence imaging were also confirmed. In fluorescence imaging experiments of zebrafish embryos, imaging of embryos was visible. The metabolic process of CDs in zebrafish with the increase of time and the fluorescence imaging of CDs could not be seen in zebrafish after 48 h, indicating that CDs could be metabolized from zebrafish. We decisively made a reasonable guess that it can be the metabolism of zebrafish the interaction between various biological molecules and CDs in the organism, or the quenching of CDs themselves. And successfully used it for biological imaging, extending the preparation method and application field of these carbon nanomaterials. To study the toxic effects of CDs on zebrafish embryo development, including hatching rate, heart rate, and survival rate of adult zebrafish, the experimental results confirm that CDs have good biological safety, which is an important reference for subsequent CDs as biomarkers.

  
  
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  Figure 1  Schematic illustration of the synthesis route of CDs

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  Figure 2  (a) TEM image (Inset: the corresponding particle size distribution map) and (b) XRD pattern of CDs

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  Figure 3  (a) XPS survey spectrum, high‐resolution XPS spectra of (b) C1s, (c) N1s, and (d) O1s of CDs

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  Figure 4  (a) UV‐Vis absorption spectrum, (b) fluorescence spectra, and (c) FTIR spectrum of CDs

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  Figure 5  Long fluorescent imaging of zebrafish: (a, e, i) bright field and (b‐d, f‐h, j‐l) fluorescent field (scale bar: 250 μm)

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  Figure 6  Metabolism of CDs in zebrafish: (a) bright field and (b‐d) fluorescent field (scale bar: 1 000 μm)

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  Figure 7  (a) Hatching rate, (b) heart rate of zebrafish embryos, (c) survival rate of adult zebrafish in CDs

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