Har Gabur is the carbide obtained from pig manure by burning. The fluorescent carbon dots (CDs) of Har Gabur were successfully synthesized through simulating the digestion process of human gastrointestinal tract. Transmission Electron Microscope (TEM) analysis showed that the average size of the prepared Har Gabur CDs was 4 nm, with good solubility in water and strong fluorescence under UV irradiation. The X-ray and Raman results showed that the Har Gabur CDs were mainly composed of oxygen “O” and carbon “C” elements, in the forms of “C=O” and “C-O.” The bond energy results showed that the nitrogen “N” atom presented as “C-N” form, which indicated that Har Gabur CDs also contain “N.” In photobleaching assay, Har Gabur CDs showed excellent light stability compared with ordinary organic dye, fluorescein, and Rhodamine B. The fluorescence intensity of Har Gabur CDs was fairly stable within a wide pH range of 3–10. When L-lysine and L-cysteine were applied for the passivation stage, the relative quantum yields were improved by 1.53 and 3.68 times, respectively. Finally, the fluorescence properties of Har Gabur CDs were tested in cells and zebrafish, illustrating that Har Gabur CD has potential in the application of biological labeling and imaging.
As one of the most important natural elements, carbon is not only involved in human body but also found to play critical role in the field of medicine [
The CD is also called carbon quantum dot (CQD). It is mainly made up of nanocarbon particles of grapheme quantum dots (GQDs) and CQDs with diameter smaller than 10 nm. The discovery of CDs is mainly through the purification of the single layer nanotube used in early preparative electrophoresis assay and the laser ablation of graphite powder and cement. The diversity of composition and structure of CDs define their different properties. The carboxyl groups on the surface of CDs enable them to have excellent water solubility and biocompatibility. In addition, the physical properties of CDs can be enhanced by chemically modifying the surface structure, for example, by applying a variety of organic, inorganic, and even biological molecules [
As an innovative fluorescence nanomaterial, CDs have palmary photoluminescence, chemical inertness, and biocompatibility [
The preparation methods of CDs have been developed and summarized into several particular refined methodologies including the synthetic, electrochemical, laser ablation, acid dissolution, and hydrothermal method [
In this study, Har Gabur CD was used for the first time as a raw material for CD preparation due to its characteristics of simple composition and cheap price. In this study, we simulate the digestion process in human body through mixing Har Gabur CDs with biological enzyme
Synthesis scheme of carbon dots derived from Har Gabur.
Muffle furnace (Beijing Cinit Electric Co., China) and U-3010 UV-visible spectrophotometer (Hitachi, Japan) were applied to the absorption measurement of carbon solution. F-2500 fluorescence spectrophotometer (Hitachi, Japan) was used for measuring fluorescence excitation and emission spectra. Zetasizer Nano (Malvern Instruments Ltd., UK) was applied for carbon hydrate particle size and surface charge determination. X-ray photoelectron spectroscopy ESCALAB 250X (Thermo, USA) was used for the determination of CD composition and C is spectrum. SpectrumGX infrared transform spectroscopy (PerkinElmer, America) and LabRAMHR800 Raman spectrometer (Joe Pingyi Feng company, France) were used to characterize the surface structure and other characteristics of carbon dots; fluorescence lifetime points by FL-TCSPC (fluorescence spectrometer Joe Feng Pingyi company, France) were measured; PHS-3C (Chengdu ark Technical Developing Company, China) was used for the acidity measurement.
0.2 g of NaCl, 0.32 g of pepsin (2330 U/mg prot, Sigma), and 0.7 ml concentrated hydrochloric acid were dissolved in distilled water up to 100 ml after adjusting pH to 1.2.
The intestinal tract liquid was prepared by dissolving 0.68 g of KH2PO4 into 25 ml distilled water. NaOH was applied for pH adjustment (pH 7.5) and additional 19 ml distilled water was added. Then, 1 g of trypsin (10010 U/mg prot, Sigma) was added, and NaOH was applied again. Distilled water was added up to 100 ml finally.
1.0 g of Har Gabur CD powder was added to a falcon flask containing prepared simulated gastric acid liquid. The falcon flask was set in the 37°C oil bath pot, allowing magnetic stirring for 12 hrs. Then, the nonreacted substances were collected through centrifugation. The supernatant was transferred into a dialysis bag (USA Viskase MWCO 1000D) and left in distilled water for 2-3 days, by changing the water once in 1 h until the pH reached neutral. After dialysis, the liquid was centrifuged at 1.6 × 102 rpm for 30 min and the supernatant was stored at 4°C. The pellets collected through gastric acid digestion were then recycled for CD particles. They were mixed with distilled water and centrifuged for 10 min at 1.6 × 104 rpm, and this step was repeated twice. The pellets were mixed with the simulated intestinal acid liquid and the flask was left in the oil bath for 12 hrs stirring. After reaction, the supernatant was repeatedly done as the above method, putting into a dialysis bag for dialysis reaction and the final liquid was stored at 4°C.
The collected supernatants from either gastric or intestinal acid liquid treatment were mixed and condensed to 25 ml. Then, the condensed liquid was microwaved for 5 min at 50 Hz. The liquid was then freeze dried and the certain amount of distilled water was added to dissolve it. The final CDs were collected through 1 min centrifugation and sent for fluorescence activity detection or stored at 4°C.
100 ml of prepared CDs liquid (0.5 mg/ml) was mixed with 1 g surface passivation agent (pH 7.25) and sent for ultrasonic treatment for 5 min. The mixture was heated up to 110°C and polylysine (PLL) and cysteine were applied for CDs surface passivation through removal of extra polylysine (PLL) and cysteine and keeping molecules of 500 Da inside of dialysis bag. Passivation using PLL was achieved through filtration. Passivation based on chitosan was also done by filtration and the extra chitosan was removed by precipitation in ethanol.
Transmission electron microscopy (TEM) was performed using a FEI Tecnai G2Spirit at an acceleration voltage of 120 kV. Nano-ZS90 Zetasizer (Malvern Instruments, Malvern, UK) was used to determine the zeta potential. Absorption and fluorescence spectra were recorded at room temperature on UV-2550 UV-Vis spectrophotometry (Shimadzu, Japan) and Luminescence Spectrometer 55 (PerkinElmer), respectively. XPS spectra were used to characterize the chemical composition using an Escalab 250 Xi X-ray photoelectron spectrometer (Thermo Scientific).
The fluorescence quantum yield was measured and calculated based on previous studies [
HepG2 cells were inoculated in 24-well culture plate (1000 cells/well). After 12 h, 1 mg/ml of prepared CDs was added to cells and cells were cultivated for extra 2 h. PBS was applied to wash cells and formalin was used for cell fixing. Cell imaging was performed through Nikon Ti-U inverted fluorescence microscope, and the excitation wavelengths were 380–420 nm and 450–490 nm for blue and green color, respectively [
Zebrafish was raised in a standardized light cycled system with a cycle of 14 h light and 10 h dark at 28°C. The imaging system for zebrafish was performed using Cri Maestro. The adult fish was cultivated into a water system with CDs (5 mg/ml) for 10 min and washed with distilled water for imaging.
The 3D image of CDs was obtained by atomic force microscopy (Atomic Force, AFM) (Figure
Direct acquisition of 3D image of C-dots by atomic force microscopy. (a) The AFM height mode image of C-dots obtained in air, the upper and bottom bars indicate the dots’ height and scale, respectively. (b) The AFM cross-sectional analysis of (a). An average hydrodynamic size of 3.6 ± 0.102 nm with a PDI of 0.422 was obtained from the DLS measurement.
UV-Vis absorption and photoluminescence (PL) emission spectra for CDs were examined. Figure
UV-Vis absorption and photoluminescence (PL) emission spectra for C-dots. (a): photograph of the C-dots excited by a UV lamp (365 nm). PL emission spectra (excitation wavelength from 325 nm to 445 nm in 20 nm increments). (b) Fluorescence image of the C-dot labeled image, its control, and bare C-dots measured with fluorescence microscope imaging system.
In order to obtain detailed structural and compositional information on the prepared carbon points, we also measured their X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS). XRD data showed that our CDs were amorphous, similar to that reported in other reports. As the wide spectrum XPS scanning shown in Figure
XPS and FTIR spectra and zeta potential. (a) The element detection. (b) XPS C1s spectrum. (c) FTIR spectra of as-prepared C-dots.
Three strong peaks at 285.4, 400, and 532.5 eV were attributed to oxygen, nitrogen, and carbon. Therefore, the main elements of the prepared CQDs are “C” (60.8%), “O” (25%), and “N” (7.2%) (see Table
X-ray photoelectron spectroscopy (XPS) spectra of the C-dots.
Name | Start BE | Peak BE | End BE | Height | CPS area (P) CPS.eV | Atomic % |
---|---|---|---|---|---|---|
C1s | 2.9 × 102 | 2.8 × 102 | 2.8 × 102 | 8.4 × 104 | 3.5 × 105 | 60.8 |
O1s | 5.4 × 102 | 5.3 × 102 | 5.2 × 102 | 1.0 × 105 | 3.6 × 105 | 25.0 |
N1s | 4 | 4 × 102 | 3.9 × 102 | 1.9 × 104 | 6.5 × 104 | 7.2 |
Si2p | 1.1 × 102 | 1 × 102 | 0.9 × 102 | 8 × 103 | 2.5 × 104 | 4.6 |
Na1s | 1.1 × 103 | 1.1 × 103 | 1.1 × 102 | 9.8 × 103 | 3.8 × 104 | 1.1 |
Cl2p | 2 × 102 | 2 × 102 | 1.9 × 102 | 1.4 × 103 | 7.8 × 103 | 0.5 |
P2p | 1.4 × 102 | 1.3 × 102 | 1.3 × 102 | 1.7 × 103 | 6 × 103 | 0.7 |
C1s | 3 × 102 | 2.8 × 102 | 2.8 × 102 | 2 × 104 | 5 × 104 | 0 |
The potential applications of the fluorescent CDs derived from plant soot were evaluated in HepG2 cells imaging. The HepG2 cells incubated with PLL-passivated CDs in Figure
Bright filed and fluorescence microphotographs of HepG2 cells incubated with PLL-passivated C-dots (C-dots) and without C-dots (Control) with an excitation filter of 450–490 nm for green color, respectively.
For organism imaging, we chose a small guppy, which is one of the vertebrates which can be used in Maestro imaging system to observe the fluorescence signal. As shown in Figure
In conclusion, through simulating human gastrointestinal absorption digestion process, we successfully synthesized highly fluorescence and biological activated carbon using gastric and intestinal juice to simply process traditional carbon black borneol. These CDs show multicolor fluorescence, excellent light stability, and their suitability used in various pH conditions. The black synthetic borneol, the dry stool carbonization of wild boars belonging to pig families, is one of the typical carbonized Mongolian medicines with the effects of invigorating the stomach and promoting the digestion and so forth. The black synthetic borneol also named Har Gabur in Mongolian is the most distinctive carbon herbs for the treatment of gastrointestinal diseases and can be mixed with other drugs used in a variety of formulations. So far, a lot of black borneol function was not clear. It needs more in-depth study and exploration.
Through the literature research we found that the current researchers have developed a variety of preparation methods of CDs, such as synthetic method, electrochemical method, laser ablation method, acid dissolution method, and hydrothermal method, in which CDs are synthesized using physical and chemical methods at the great costs of labor force and money. However, an important preparation method, enzyme digestion method, has not yet been applied to the preparation of CDs. More importantly, there is close relationship between this method and traditional medicine digestion in the human body and carbon absorption in the human body. Thus prepared CDs completely dissolve in the human body without any security risks and even do good to improve immunity and have other health effects.
In enzyme digestion method, Har Gabur was processed by intestinal fluid to prepare CDs. Following the heat treatment in 37°C water, pepsin or trypsin will first attack the defect area of the surface of the carbon particles, “cutting” or “dividing” those carbon particles, forming the smaller size quantum dots, ice sheet fluorescent CDs.
The surface of ice sheet fluorescent CDs prepared by such method is rich in amino functional organic groups, which makes it not only have good dispersibility, water solubility, easy functionalization, and biological coupling markers but also have good biocompatibility. We will model ice sheet fluorescent nano-CDs to explore the relationship between chemical properties and biological properties of nanomaterials in the field of cells. Choosing the most suitable carbon for preparation and biological safety detection is also our next plan. It will help to carry out the new fluorescent nanomaterials development and application of optical imaging studies. Ice sheet fluorescent CDs provide valuable information for optical imaging in tumor diagnosis, especially early diagnosis and imaging research.
The authors declare that they have no conflicts of interest.
Jun Ai provided technical support.
This work was supported by the project “The Innovative Research Team of the New Drug Research and the Industrialization of Mongolian Medicine” of “Prairie Talents” Engineering, no. NEI ZU TONG ZI