Natural Carbon Nanodots: Toxicity Assessment and Theranostic Biological Application

This review outlines the methods for preparing carbon dots (CDs) from various natural resources to select the process to produce CDs with the best biological application efficacy. The oxidative activity of CDs mainly involves photo-induced cell damage and the destruction of biofilm matrices through the production of reactive oxygen species (ROS), thereby causing cell auto-apoptosis. Recent research has found that CDs derived from organic carbon sources can treat cancer cells as effectively as conventional drugs without causing damage to normal cells. CDs obtained by heating a natural carbon source inherit properties similar to the carbon source from which they are derived. Importantly, these characteristics can be exploited to perform non-invasive targeted therapy on human cancers, avoiding the harm caused to the human body by conventional treatments. CDs are attractive for large-scale clinical applications. Water, herbs, plants, and probiotics are ideal carbon-containing sources that can be used to synthesize therapeutic and diagnostic CDs that have become the focus of attention due to their excellent light stability, fluorescence, good biocompatibility, and low toxicity. They can be applied as biosensors, bioimaging, diagnosis, and treatment applications. These advantages make CDs attractive for large-scale clinical application, providing new technologies and methods for disease occurrence, diagnosis, and treatment research.


Introduction
The materials studied in the past were at the micrometer scale; however, in the past few decades, nanometer-scale development has changed, which has become an essential direction of scientific and technological development. Nano-sized materials are widely used in various fields, such as medicine, biosensor development, energy research, and catalysis. As fluorescent nanomaterials can emit light, they have the potential for application in biomarking technology, such as semiconductor-based quantum dots containing cadmium sulfide (CdS) or cadmium selenide (CdSe). However, most materials with a high quantum yield (QY, φ) on the market include heavy metal components, which pose risks related to their biological toxicity and cytotoxicity. Contamination may occur during the synthesis process, and they are not adequately recycled after use; as such, the associated harm to the environment should not be underestimated. The above shortcomings limit the application range as semiconductor quantum dots.
In recent years, nanotechnology has been widely used in the field of biomedicine [1]. Generally, when the scale of a substance drops to the nanometer level, its physical and 1.
Imaging: multicolor fluorescent images of mammalian cells, plant cells, and microorganisms, and imaging in mice; 2.
Photocatalysis: the degradation of organic molecules, the reduction in CO 2 , and water splitting; 3.
Sensors: food quality and safety, drug analysis, environmental pollution determination, immunoassay, and other fields, such as detecting heavy metal ions, anions, pesticides, molecules, small organic molecules, and/or nucleic acids; 5.
Electrocatalysis: mainly used in oxidation-reduction reactions, oxygen evolution reactions, hydrogen evolution reactions, and reduction reactions for carbon dioxide, and dual-function catalysts; 6.
Biomedicine: photodynamic therapy, photosensitizers for cancer cell destruction, radiotherapy, the tracing and delivery of drugs or genes, drug release, and anticancer drugs. Most carbon sources derive from exhausted petroleum resources or non-enviro mentally friendly manufacturing processes. Therefore, using recycled materials and b renewable resources to develop high-performance CDs is a critical green environmen issue. In recent years, the awareness of environmental protection has significantly ris and, as such, naturally derived CDs have gradually received more attention.
The main factor is that they are derived from renewable and sustainable biologi materials; for example, lignocellulose from dead wood, waste wood, rice straw, bagas wheat straw, etc. Suppose that such a raw material can be used as a carbon resource a converted into CDs with therapeutic and diagnostic value. In that case, we can imag the result will not overwhelm the demand of the food supply chain. Still, it can also a dress waste disposal problems while applying the resource to produce high value-add products such as electronics, energy, and biomedicine. Table 1 summarizes the existi research on converting various natural resources into CDs, and the associated applicatio and emission spectra are described in detail. Most carbon sources derive from exhausted petroleum resources or non-environmentally friendly manufacturing processes. Therefore, using recycled materials and bio-renewable resources to develop high-performance CDs is a critical green environmental issue. In recent years, the awareness of environmental protection has significantly risen, and, as such, naturally derived CDs have gradually received more attention.
The main factor is that they are derived from renewable and sustainable biological materials; for example, lignocellulose from dead wood, waste wood, rice straw, bagasse, wheat straw, etc. Suppose that such a raw material can be used as a carbon resource and converted into CDs with therapeutic and diagnostic value. In that case, we can imagine the result will not overwhelm the demand of the food supply chain. Still, it can also address waste disposal problems while applying the resource to produce high value-added products such as electronics, energy, and biomedicine. Table 1 summarizes the existing research on converting various natural resources into CDs, and the associated applications and emission spectra are described in detail.

Toxicity Evaluation of Natural Carbon Nanodots
CDs can be derived from a wide range of synthetic raw materials, low-cost, stable chemical properties, and non-toxic materials. The application of CDs in medicine and pharmacy was recently extensively studied. One of the most promising nanomaterials is carbon quantum dots (CQDs). For this purpose, the toxicity of CQDs was investigated in cells and living systems ( Table 2). In 2019, L. Janus used human dermal fibroblasts to conduct a cytotoxicity study of N-doped chitosan-based CDs [129]. As shown in Figure 2, after 48 h, the cell viability was recorded as 94%. CDs were synthesized by utilizing non-toxic raw materials and removing unreactive residues during purification.   First, we start with the selection of carbon source and the adjustment of dopants that synthesize carbon dots, and at the same time list the test models of carbon dots. Secondly, according to the selective response of the carbon dot to the biological models, the corresponding toxicity was constructed, and the actual application was investigated. As mentioned in Table 2, CQDs are usually doped with nitrogen to enhance their fluorescence quantum yield and optical performance [130,131]. CDs doped with nitrogen (such as carbon nanotubes, graphene, hollow spheres, etc.) have unique properties. They can inject electrons into the carbon substrate to change the electron and transport characteristics such as sensors, nanogenerators, etc., and are widely used and have become a research trend. Moreover, green methods that utilize natural biomass/biowaste and micro-organisms without introducing toxic chemicals as CDs precursors have been widely used to synthesize CQDs [132].  S. Cong et al. [122] used PC12 cells for a cytotoxicity study of CQDs obtained from a roast duck. After 36 h, the cell viability of PC12 was recorded as 91.19% at the concentration of 1 mg/mL. In addition, using CDs at a concentration of 15 mg/mL for treating nematodes did not lead to any death for 24 h. These results indicate the low toxicity of CDs, even after a long period of exposure at high concentrations. In 2021, R. Atchudan et al. [141] used C. elegans as an animal model for their toxicity evaluation of CQDs. As displayed in Figure 3, the CQDs synthesized from banana peel were shown to have low nematode toxicity, even at a high concentration of 200 ug/mL. These results can be explained by their utilizing non-toxic raw materials without adding any passivates or additives. tion of 1 mg/mL. In addition, using CDs at a concentration of 15 mg/mL for treating nematodes did not lead to any death for 24 h. These results indicate the low toxicity of CDs, even after a long period of exposure at high concentrations. In 2021, R. Atchudan et al. [141] used C. elegans as an animal model for their toxicity evaluation of CQDs. As displayed in Figure 3, the CQDs synthesized from banana peel were shown to have low nematode toxicity, even at a high concentration of 200 ug/mL. These results can be explained by their utilizing non-toxic raw materials without adding any passivates or additives.

Theranostic Application of Natural Carbon Nanodots
Therefore, fluorescent materials are expected to be critical in biological applications. CDs have become the focus of attention as new nanomaterials due to their excellent light stability, fluorescence, good biocompatibility, and low toxicity. Surgery, radiotherapy, and chemotherapy are inevitable treatments for most cancer patients, but these processes have considerable side effects on the human body. However, fluorescent nanomaterials have the advantages of high fluorescence stability, low biotoxicity, and good biocompatibility. The most important thing is that they can be used to perform non-invasive targeted therapy on human lesions, exploiting their characteristics to avoid harm to the human body caused by the abovementioned treatments.
This paragraph covers the luminescence mechanism of CDs and their applications in biology, focusing on applying natural CDs in biological diagnosis and treatment. We discuss the combination of CDs with specific targeting molecules to form CD-based probes for detecting fluorescent signals. With the help of advanced optical imaging technology, real-time dynamic monitoring of molecules in cells and organisms can be carried out, and rapid immunofluorescence analysis of primary infectious disease sources can be carried out. They can provide new technologies and methods for disease occurrence, diagnosis, and treatment research [150].

Bioimaging
The discrete and diverse microstates of CDs lead to broad excitation and emission ranges [151,152]. CDs have many unique properties, and their excellent photostability can provide fluorescent information in a biological environment. The surface modification of functional groups can make CDs more helpful in applying biomarkers (Table 3). In this section, we compare bioimaging from cells to living animals, emphasizing the biological diversity to prove the generalized safety of CDs. In cell imaging applications, CDs are usually applied to HepG2 cells, HeLa cells, T24 cells, and so on [27,102,120,124]. In addition, normal cell lines have been used in trials, showing good cell compatibility [24,71]. Alam et al. treated HaCaT cells with cabbage-derived CDs and showed that, at 500 µg/mL of CDs, the cell viability was 100%. Furthermore, CDs' tunable excitation and emission show promise for normal cell imaging under the irradiation of confocal fluorescence microscopy [71]. In a cell imaging experiment, Mehta et al. considered CDs originating from apple juice and three fungal (M. tuberculosis, P. aeruginosa, and M. oryzae) sources. Cell compatibility was shown by feeding more than 100 mg/mL of CDs. The germination of M. oryzae spores also strongly indicated how CDs are good biocompatible nanocomposites at high concentrations (i.e., 400 mg/mL). The fungi appeared red, green, and blue in confocal laser microscopic images [62]. Kasibabu  incubating C. elegans in NCDs for 24 h from 0 to 200 μg/mL (Figure 4a-f) [96]. Cong et a using roast duck as the source and pyrolyzing at 200 °C , 250 °C , and 300 °C , synthesize single-fluorescence CDs. C. elegans were treated with 15 mg/mL of the CDs pyrolyzed 300 °C (300-CDs) for 24 h. Compared with the wild-type group, the accumulation an uptake of 300-CDs made the intestine appear blue under UV light exposure (Figure 4g- [122]. The murine model has also been widely used for determining the efficiency of CD   [96], ACS Publications, 2018. Bright-field (g) wild-type C. elegans imaging and (i) 300-CDs treated C. elegans imaging; UV-exposed (h) wild-type C. elegans imaging and (j) 300-CDs treated C. elegans imaging, adapted with permission from [122], Elsevier, 2019.
They concluded that, with the assistance of PL, Fn-CDs show strong fluorescence in the bladder and are eliminated after 7 h (Figure 5d) [28]. Therefore, CDs can perform well as imaging nanoparticles and be adapted to different cell lines and living animals. In addition, their multiple emission ranges, low toxicity, and small size confer their high potential in future clinical applications.  They concluded that, with the assistance of PL, Fn-CDs show strong fluorescence in the bladder and are eliminated after 7 h (Figure 5d) [28]. Therefore, CDs can perform well as imaging nanoparticles and be adapted to different cell lines and living animals. In addition, their multiple emission ranges, low toxicity, and small size confer their high potential in future clinical applications.

Sensors
Inorganic ions are critical for creatures, not only for enhancing the efficiency of catalytic reactions in bio-systems but also for maintaining our fundamental life functions. However, it is harmful to have too many metal cations, which are highly toxic to human beings. Therefore, developing sensors for inorganic ions is a simple beneficial method to collate the concentration and standard. Owing to the multiple PL of CDs, we can observe the intensity variation and chelation quenching effect at other peaks in the PL image. For natural CDs sensors, Fe 3+ , Hg 2+ , and Cu 2+ are the most examined targets. Shen et al. assessed HepG2 and HeLa cell images after incubating with CDs and Fe 3+ .
According to the decreased blue fluorescence under 405 nm irradiated light, Fe 3+ had a practical quenching effect on the CDs (Figure 6a-d) [33]. Hu et al. prepared double-emitted biomass nitrogen co-doped CDs (B-NCdots) for Cu 2+ probing in T24 cells. Similarly, the quenching effect was still available for Cu 2+ , causing a decreased intensity of blue

Sensors
Inorganic ions are critical for creatures, not only for enhancing the efficiency of catalytic reactions in bio-systems but also for maintaining our fundamental life functions. However, it is harmful to have too many metal cations, which are highly toxic to human beings. Therefore, developing sensors for inorganic ions is a simple beneficial method to collate the concentration and standard. Owing to the multiple PL of CDs, we can observe the intensity variation and chelation quenching effect at other peaks in the PL image. For natural CDs sensors, Fe 3+ , Hg 2+ , and Cu 2+ are the most examined targets. Shen et al. assessed HepG2 and HeLa cell images after incubating with CDs and Fe 3+ .
According to the decreased blue fluorescence under 405 nm irradiated light, Fe 3+ had a practical quenching effect on the CDs (Figure 6a-d) [33]. Hu et al. prepared double-emitted biomass nitrogen co-doped CDs (B-NCdots) for Cu 2+ probing in T24 cells. Similarly, the quenching effect was still available for Cu 2+ , causing a decreased intensity of blue and green, as shown in Figure 6e,f [117]. Furthermore, bio-related molecules, including peptides and drug-containing cells, are even more crucial. Some researchers designed chemically sensitive CDs to assist in directly resolving the effects of molecules by monitoring the decrease or increase in fluorescence. Liang et al. added 0.5 mM and 1 mM of glutathione and 150 µg/mL rose-red fluorescence CDs (wCDs) in L929 cells, HeLa cells, and HepG2 cells. An intense quenching effect of glutathione was observed in L929 cells and HeLa cells. However, no apparent variation occurred in HepG2 cells, which implies a distinct response of wCDs to glutathione (GSH) in various cells (Figure 7) [24]. anticancer medicine, by taking advantage of the PL of plum-based carbon quantum dots (PCQDs). The dual-emitted property at the wavelengths of 491 nm and 591 nm provided a ratiometric calibration curve as a function of the DOX concentration. They also confirmed the accuracy by analyzing urine and serum samples (Figure 8b) [27]. CDs are suitable for detecting different kinds of molecules and ions. The intensity changed at a single emission peak, but the amplitude ratio of two emission peaks is valid for sensing experiments. Due to their intrinsic properties, CDs show promise in the bio-sensing field and are applied to cancer therapy.  The dual-emitted property at the wavelengths of 491 nm and 591 nm provided a ratiometric calibration curve as a function of the DOX concentration. They also confirmed the accuracy by analyzing urine and serum samples (Figure 8b) [27]. CDs are suitable for detecting different kinds of molecules and ions. The intensity changed at a single emission peak, but the amplitude ratio of two emission peaks is valid for sensing experiments. Due to their intrinsic properties, CDs show promise in the bio-sensing field and are applied to cancer therapy.

Antibacterial Activity
Bacteria are well-known as the origins of various diseases. Recently, super bacteria have appeared globally, which cause incurable illnesses due to the abuse of antibiotics. In addition, people have come to pay more attention to the side effects of antibiotics and wish to avoid unexpected risks. Nanomedicines, especially CDs, have been taken into consideration as substitute methods. E. coli and S. aureus are the most common types of bacteria for investigating how nanomedicines or antibiotics work to induce apoptosis in bacteria. Wang et al. used CDs (ACDs) derived from Artemisia argyi leaves to treat E. coli and S. aureus cultures. According to the SEM images (Figure 9a-h), it can be seen that the cell walls of E. coli were destroyed; however, there was no distinct difference between treated and untreated S. aureus. This means that ACDs are selective to Gram-negative bacteria due to the structural properties of their cell walls [123]. Sun et al. synthesized chlorhexidine gluconate CDs from large to small (l-CGCDs, m-CGCD, and s-CGCDs) to determine the relationship between size and antibacterial activity. From the SEM imagery (Figure 9i), it can be seen that the rigidity of cell walls was the strongest in the control group and decreased from l-to s-CGCDs groups. These results revealed that CGCDs lead to frustration in the walls and membranes of E. coli and S. aureus. Bacterial death can be controlled by tuning the size of the CGCDs [76]. Ma et al. tested three kinds of CDs, including osmanthus leaves-derived CDs (OCDs), milk vetch-derived CDs (MCDs), and tea leaves-derived CDs (TCDs). In Figure 10, 80% of E. coli and S. aureus were killed by OCDs at a 1 mg/mL concentration, while 70% of bacteria survived in the MCDs group. In addition, E. coli had stronger resistivity than S. aureus among these CDs. CDs are internalized into bacteria. The outer surface of bacteria is attached to CDs leading to indirect proliferating inhibition [153][154][155]. These results prove the natural sources are essential for the synthesis of CDs [79]. As mentioned above, the tunability of raw material and diameters primarily affect the antibacterial efficiency and selectivity of the resultant CDs. Treating the patient's wounds after surgery with CDs with editable properties can tremendously decrease the associated risks.

Antibacterial Activity
Bacteria are well-known as the origins of various diseases. Recently, super bacteria have appeared globally, which cause incurable illnesses due to the abuse of antibiotics. In addition, people have come to pay more attention to the side effects of antibiotics and    [123], Royal Society of Chemistry, 2020. SEM images of (i) E. coli and S. aureus untreated and treated with 75 µg/mL and 50 µg/mL of s-CGCDs, m-CGCDs, and l-CGCDs in Luria-Bertani broth medium for 6 h, adapted with permission from [76], Elsevier, 2021.

Anticancer Activity
At present, cancers are prevalent within all age ranges. Cancers may be fatal due to unexpected syndromes as well as the disorder of living functions. Furthermore, conventional cancer therapies are long-term processes. Surgeries are straightforward methods, but recovery typically poses a challenge for patients. Even though chemotherapy seems safer, the currently used drugs lack selectivity and affinities to specific tumors. Some targeted therapies have been developed in recent years. However, they are expensive and only valid for certain types of cancers. CDs can provide great theranostic nanomedicines in cancer treatments. Scientists have attempted to eliminate cancer cells through photothermal therapy (PTT) [81,82] and photodynamic therapy (PDT) [83,87,129] to fulfill tumor targeting. Li et al. tested NIR-II emitted (900-1200 nm) CDs (CDs), adapted for 808 nm laser photothermal therapy. According to the in vivo test (Figure 11), the temperature increased to 50 • C in the intratumoral environment after intravenous injection. Tumor inhibition and volume shrinkage were observed within 6 days, compared with the PBS group. No detectable damage to tissues or weight loss after the treatment confirmed the high biocompatibility of the CDs [156]. Jia et al. prepared red-light absorbing (610 nm) CDs from Hypocrella Bambusa (HBCUs). They found that HBCDs highly generate 1 O 2 under 635 laser irradiation. The reactive radicals induced apoptosis of cancer cells, which is helpful in the hypoxia tumor environment.
As shown in an in vivo experiment (Figure 12), due to the synergistic effect of PDT and PTT, the temperature at the tumor site increased to 56.4 • C in 10 min. Secondly, a good tumor inhibition effect was found after 14 days of therapy, even though the tumor could not be depleted thoroughly. No harmful phenomena were observed in other organs, indicating the safety of the treatment [82]. Xue et al. conducted modification with polyethylene glycol diamine (H 2 N-PEG-NH 2 ), chlorin e6 (Ce6), and transferrin (Tf) on natural biomass CDs (NBCDs) to increase the targeting efficacy.

Anticancer Activity
At present, cancers are prevalent within all age ranges. Cancers may be fatal due to unexpected syndromes as well as the disorder of living functions. Furthermore, conventional cancer therapies are long-term processes. Surgeries are straightforward methods, but recovery typically poses a challenge for patients. Even though chemotherapy seems safer, the currently used drugs lack selectivity and affinities to specific tumors. Some targeted therapies have been developed in recent years. However, they are expensive and Figure 10. Graphs of E. coli and S. aureus incubated with varying concentrations of OCDs, TCDs, and MCDs for 24 h; alive ratio of E. coli and S. aureus calculated using UV-Vis spectroscopy methods, adapted with permission from [79], Elsevier, 2020. CDs from Hypocrella Bambusa (HBCUs). They found that HBCDs highly generate 1 O2 u der 635 laser irradiation. The reactive radicals induced apoptosis of cancer cells, which helpful in the hypoxia tumor environment.  The resulting products, NBCD-PEG-Ce6-Tf, were shown to remain within the tumor environment for 120 h using a real-time NIR fluorescence image (Figure 13a). The mice were irradiated daily under a 650 nm laser to generate 1 O 2 . During the 21-day process, tumor growth was stopped, and the tumors were ablated, indicating no conflict between the NBCDs and modifications (Figure 13b cells. The presence of orange colors and blue dots indicate cell fragmentation due to CDs (Figure 14d-g) [80]. CDs have a powerful potential in the anticancer field. Their flexibility is because the CDs are modified with various molecules, which can improve the uptake by tumor cells and increase the tumor-killing ability of the nanohybrids. The resulting products, NBCD-PEG-Ce6-Tf, were shown to remain within the tumor environment for 120 h using a real-time NIR fluorescence image (Figure 13a). The mice were irradiated daily under a 650 nm laser to generate 1 O2. During the 21-day process, tumor growth was stopped, and the tumors were ablated, indicating no conflict between the NBCDs and modifications (Figure 13b,c) [126]. Li et al. synthesized reactive oxygen species (ROS)-generating CDs from ginger. The CDs were harvested from HepG2 tumor inoculating mice; next, the tumor regressions were observed in the C-dot (440 μg) treated group; the tumor growth was prominently delayed, which attained only 3.7 ± 0.2 mg. In contrast, tumors in the PBS group grew up to 104 mg [84]. Boobalan et al. added 30 μg/mL of CDs into P. aeruginosa. They observed the destruction of cell walls due to ROS attack, in agreement with the results of ROS fluorescence detection using a fluorogenic dye, 2′,7′dichlorofluorescein diacetate (DCFDA) (Figure 14a-c). MDA-MB-231 breast cancer cells were treated with CDs (3.34 μg/mL). Cell apoptosis staining, acridine orange and ethidium bromide (AO/EtBr), and nuclear staining (Hoechst 33342) were applied to the cells. The presence of orange colors and blue dots indicate cell fragmentation due to CDs (Figure 14d-g) [80]. CDs have a powerful potential in the anticancer field. Their flexibility is because the CDs are modified with various molecules, which can improve the uptake by tumor cells and increase the tumor-killing ability of the nanohybrids.

Discussion and Conclusions
Carbon nanomaterials have been widely used in various scientific, engineering, and commercial fields, due to their high catalytic activity and good stability. Among them, the new "zero-dimensional" carbon nanomaterials, CDs, have unique optical properties, such as stable fluorescence signals, no light scintillation, adjustable excitation and emission wavelength, and low biological toxicity and biocompatibility. These advantages gradually led to the popularity of researching carbon nanomaterials, widely used in bioimaging, natural cell labeling, sensors, photocatalysis, solar cells, and light-emitting elements. This

Discussion and Conclusions
Carbon nanomaterials have been widely used in various scientific, engineering, and commercial fields, due to their high catalytic activity and good stability. Among them, the new "zero-dimensional" carbon nanomaterials, CDs, have unique optical properties, such as stable fluorescence signals, no light scintillation, adjustable excitation and emission wavelength, and low biological toxicity and biocompatibility. These advantages gradually led to the popularity of researching carbon nanomaterials, widely used in bioimaging, natural cell labeling, sensors, photocatalysis, solar cells, and light-emitting elements. This article mainly reviewed the different synthesis methods of CDs (including top-down and bottom-up methods) and their applications. Their luminescence properties can be adjusted through surface modification. They have been applied in many fields and have great potential. The function of CDs can also be modified by using various surface functional groups, allowing them to act, for example, as detectors and cleaners for different heavy metal ions or by doping with other ions. By controlling the surface light energy groups, they can be better used in the required fields.
Green chemistry is a discipline that has gradually received attention in recent years. The core concept focuses on the development of environmentally friendly chemical technologies. At the technical level, chemical technologies and methods are applied to reduce or eliminate the use and generation of hazardous substances in chemical synthesis and analysis, and recovery and reuse technologies are combined with increasing energy and material use efficiency. Green chemistry and nanotechnology have become emerging technology research and development directions in recent years. With the deepening of the concept of sustainability, combining the advantages of the two and accelerating the expansion of their research and development applications has become a top priority. Laboratories are committed to determining the complementary relationships between green chemistry practices and nanotechnology and applying them to materials development, chemical analysis, energy, environmental, and other related fields. The preparation of CDs well embodies the concept of green chemistry. Cheap, environmentally friendly carbon source precursors and natural renewable raw materials as carbon sources for preparation, such as eggs, grass, leaves, silk, coffee grounds, beer, and other materials, have become carbon sources for the synthesis of CDs.
Excellent performance and a unique structure provide natural CDs unlimited charm and various changes. Natural CDs, combined with biological and pharmaceutical molecules of interest through surface modification, seem to be an emerging platform for imaging probes that are both diagnostic and therapeutic. The next generation of nano-molecular probes integrates a variety of fluorescent dyes, drugs, and multifunctional nanomaterials into a single nanoprobe, providing superior signal contrast, controllable transmission, and targeted drug delivery capabilities. However, before this kind of multifunctional imaging probe was used in diagnosis and treatment, there were still many challenges, such as long-term safety, risk-benefit, biocompatibility, and biodistribution, to be evaluated. In the future, we need to solve several critical scientific problems in the research of natural CDs. First of all, the uncertain chemical groups on the surface indicate that natural-synthetic CDs are a kind of unsure material. It means the method of natural mass production of high-quality CDs is still a big challenge. Secondly, due to the different sources of naturally transformed CDs, the luminescence centers of CDs are also dissimilar. Finding a suitable luminescence position is also essential to research content. Third, categorizing different natural CDs and conducting a systematic comparative analysis will be beneficial research methods. Nanotoxicology is the emerging study of potential adverse effects derived from the interaction between nanomaterials and biological systems, and it is bound to become more critical. To further clarify its physical toxicity and adjust the size and structure accordingly, it will significantly improve CDs' application performance while expanding a more comprehensive range of applications. The scale and complexity of biomedical issues have always been an enormous challenge for researchers. It is more necessary to conduct cross-disciplinary research in chemistry, physics, materials, biomedical engineering, toxicology, public health, and clinical medicine. As more research on natural CDs continues to develop, the topic will achieve breakthroughs and progress in a short period.