Cellulose hydrogel is a novel carbon-source and doping-material-carrier to prepare fluorescent carbon dots for intracellular bioimaging
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Carbon dots (CDs) is considered as a potential candidate for biological labeling due to its excellent biocompatibility, and element-doping was usually used to improve its labeling brightness. Thus, the carbon source with material-carried function will be of importance to produce the element-doped CDs. In present work, cellulose hydrogel was used both as the carbon source and the doping-material-carrier to obtain the N-doped CDs. The groups in so-obtained CDs were measured by means of ultraviolent-visible spectrophotometer (UV–Vis), Fourier infrared spectroscopy (FT-IR), and X-ray photoelectron spectroscopy. The microstructure of CDs was observed by means of high-resolution transmission electron microscope. The fluorescence quantum yield (QY) of the CDs was detected, and the labeling brightness on living cells was subsequently investigated. Finally, the influence of element-doping on the cytotoxicity was measured. The experimental results showed that Cellulose hydrogel is a nice carbon source and doping-material-carrier to produce fine N-doped CDs due to its distinct spatial network structure. The carried NaOH and urea in cellulose hydrogel significantly improved the QY of the CDs from 0.0542 ± 0.0030 to the maximum 0.1965 ± 0.0013. For the same kind of CDs, the smaller the particle size is, the higher the QY value is. The CDs immobilized in the cytoplasm of the living cells, and contributed to a non-specific fluorescent labeling. The labeling brightness is both the QY value of the CDs and the uptake rate of the living cells dependent. For the same cell line, the QY of CDs and the labeling brightness of living cells are significantly linear correlated. The cytotoxicity of CDs is low enough for a long-time observation on living cells.
KeywordsCarbon dots Cellulose hydrogel Quantum yield Labeling brightness Living cells
Carbon quantum dots (CDs), a new zero-dimensional nanomaterial, has advantages on the stable photoluminescence, excellent biocompatibility, and low cost when compared to those traditional semiconductor quantum dots (CdS, CdSe, CdTe, et al.). Thus, CDs is regarded as a potential candidate for application in ion/molecule detection, biosensors, bio-imaging, especially in living cell imaging [1, 2, 3].
A good fluorescent probe in living cells imaging should be low toxicity, high stability, bright fluorescence, good water solubility, and sometimes specific binding. As a fluorescent probe, CDs was demonstrated that it can be internalized into the cells to mark the cytoplasm and the membrane . Different types of cells have different responses on CDs labeling, thus, CDs was considered to distinguish cancer cells from those normal cells. For example, CDs combined with folic acid was produced to distinguish between cancer cells and normal cells . Receptor-mediated endocytosis of CDs provides a more accurate and selective method for cancer diagnosis . However, the labeling brightness of CDs needs to be further improved, and the specific labeling property of CDs needs to be developed .
Element-doping is an effective way to improve such labeling properties as brighter imaging and specific labeling because it can introduce helpful groups to CDs. Labeling brightness is a key index for a fluorescent probe. To improve the labeling brightness of CDs and thus enables it to be more effectively used, such methods as passivation and elements doping were tried to modify the fluorescent properties of CDs . For example, Nitrogen (N) doping can effectively accelerate the rate of electron transfer in the molecule without significantly increasing the size of CDs , thus, N-doped CDs exhibits a brighter labeling image. Phosphorus is also a non-metallic heteroatom with high electron-donating ability. The new active sites provided by phosphorus doping can improve the electron transfer ability . Specific labeling is another property to approach, and element-doping can provide groups such as –NH2, –COOH, etc. to bonding the target antibody or nucleic acid aptamer .
For the doping-element-modified CDs, the doping dose, doping yield, and doping methods need to be considered besides the doping materials. Normally, the mixture of the doping material and the carbon material were used to produce the modified CDs. Biomass raw materials, such as chitosan , waste wood chips  are more and more widely used due to its low cost, natural non-toxicity, and abundant sources. Cellulose, the most abundant reserved biomass raw material on the earth, has been successfully applied in the fields of drug delivery, bio-imaging, catalysis and sensing [13, 14] due to its excellent degradability, renewable and bio-friendliness. As a carbon source, cellulose powder has also been used to synthesize CDs through high-pressure homogenization . To improve the labeling brightness of CDs, cellulose-based biowaste of willow catkin was used to yield nitrogen and sulfur co-doped CDs (N/S-CDs) via the one-step combustion . Usually, the mixture of cellulose and doping element contained chemicals in a state of solid powder were used as the raw materials to produce the modified CDs . However, the uniformity and dispersity of the doping material need to be considered because it may have influence on the modification effect.
Compared to cellulose powder, the spatial network structure of cellulose hydrogel makes it be a better doping-material-carrier, and it has already been used in drug delivery. Thus, in present work, cellulose hydrogel was used as both the carbon source and the doping-materials-carrier to produce the N-doped CDs. The fluorescent properties of so-obtained CDs were characterized to confirm that whether the cellulose hydrogel system is competent to yield the element-doped CDs. In additional, the labeling properties of so-obtained CDs on living cells were quantitatively studied to find the correlation between the quantum yield (QY) of CDs and the imaging brightness. Finally, the cytotoxicity of so-obtained CDs was measured to see whether the N-doping will affect the biocompatibility of CDs.
2 Materials and methods
2.1 Materials and reagents
Cotton staple cellulose (Mw = 8 × 104) was obtained from Hubei Chemical Fiber Group Co., Ltd., China. Epichlorohydrin (ECH), NaOH and urea (analytically grade) were purchased from Sinopharm Group, China. Human Amniotic Epithelial Cells and the cervical cancer cell line Hela cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, China. Fetal bovine serum (FBS), MEM culture medium, PBS buffer 0.25% trypsin (containing EDTA) were supplied by Gino Biomedical Technology Co., Ltd, China. Cell Proliferation Cytotoxicity Detection Kit (CCK-8), ER-Tracker Red, Phalloidin TRITC and dialysis bags were purchased from Beyotime Biotechnology Co., Ltd., China. Millipore water was used for the entire experiment.
2.2 Synthesis of the cellulose hydrogel and CDs
The aqueous solution containing 7 wt% NaOH, 12 wt% urea and 81 wt% H2O was precooled to − 20 °C. Then cellulose was added to this solution, the mixture was stirred to dissolve the cellulose and finally form a 4% (wt%) transparent cellulose solution. Subsequently, a certain amount of ECH was dropped into the cellulose solution and incubated at 60 °C for 4 h to form the chemical cross-linked cellulose hydrogel . After that, the cellulose hydrogel was rinsed in deionized water to thoroughly remove residual reagents to get the pure cellulose hydrogel.
To investigate the influence of alkali and urea on the QY and cell labeling property of CDs, the prepared cellulose hydrogel was treated in different ways before being used as the CDs source: (1) The prepared raw hydrogel was directly used to produce CDs without any treatment. In this case, “Cellulose CDs” was obtained. (2) The pure cellulose hydrogels were immersed in 7 wt% NaOH solution, 12 wt% urea aqueous solution, and 7 wt% NaOH plus 12% urea solution, respectively for 48 h and the CDs produced from these different ways were marked as “NaOH/cellulose CDs”, “urea/cellulose CDs”, and “NaOH/urea/cellulose CDs”, respectively. The above 4 kinds of CDs were produced by one-step hydrothermal method, and the cellulose hydrogels in Teflon-lined stainless-steel autoclaves were incubated at 200 °C for 12 h. After centrifugation at high speed and ethanol extraction, the CDs with different particle sizes were collected via gradient dialysis (the cut-off molecular weights of dialysis bags were 14000D, 8000D, 3500D and 1000D). The CDs with different size were identified as the “> 14000D CDs” “8000D–14000D CDs” “3500D–8000D CDs”, “1000D–3500D CDs” and “< 1000D CDs”.
The UV–visible absorption test was performed on a CARY-100 UV–Vis spectrophotometer (Agilent, USA) with a scan range of 200–800 nm for structural analysis of CDs.
To study the structure and functional groups of the CDs, (FT-IR) spectrometer (Nicolet Instruments, USA) was employed to obtain the FT-IR spectra by using a potassium bromide tableting method. X-ray photoelectron spectroscopy (XPS) spectra were recorded with ESCALAB 250XI (Thermo, USA).
The fluorescence spectra of the CDs were obtained by using a Hitachi F-2500 fluorescence spectrophotometer (Hitachi, Japan) with a scan range of 320–600 nm at a scan rate of 1500 nm/min.
JEM-2100F high-resolution transmission electron microscopy (HRTEM, Japan) was used to observe the morphology and microstructure of the fluorescent CDs.
2.4 Quantum yield
2.6 Cell imaging experiments
AECs and Hela cells were seeded in the confocal petri dishes with a density of 5 × 104/cm2 and incubated in the 37 °C, 5% CO2 incubator for 24 h. Then, the old medium was replaced by the fresh medium that containing 500 μg/mL CDs and further cultured for another 4 h. After being rinsed with PBS for 3 times (10 min for each time), the cells were immediately observed under the laser confocal microscope (LEICA TCS SP5, German) at the exciting wavelength of 405 nm. In the case that the cells co-stained with the endoplasmic reticulum, the CDs-incubated cells were first rinsed with HBSS twice and incubated in 1 μmol/L ER-Tracker Red for 30 min at 37 °C. In the case that the cells co-stained with f-actin, CDs-cultured cells were fixed with 4% paraformaldehyde, then treated with 0.2% Triton-X, and finally incubated in 1 μmol/L Phalloidin TRITC for 1 h at room temperature . All fluorescent images of CDs labeling were recorded under the same camera parameters (scan area: 512 × 512; scanning speed: 400 Hz; pinhole size: 1 Airy) and the fluorescent brightness was quantitatively analyzed from these original images by means of Image J.
2.7 Data statistics analysis
All data are presented as the Mean ± SD, and the statistical analysis was performed by using SPSS 19.0. The statistical significances between data sets were expressed as p value or “Sig.”. P or “Sig.” < 0.05 was considered as statistically different, and P or “Sig.” < 0.01 was considered as extremely significant in difference. The one-way ANOVA analysis was used for multiple comparisons and Sig. < 0.05 was considered as a significant difference. The Pearson correlation coefficient analysis was used to describe the correlation between the two variables. The closer the Pearson value to 1 is, the higher the correlation is.
3 Experimental results
3.1 Characterization of CDs
The FT-IR spectra shown in Fig. 1b revealed more information about the groups in the CDs. Peaks in the range of 3000–3500 cm−1 correspond to stretching vibration of O–H or N–H. The peak at 1431 cm−1 corresponds to the telescopic vibration peak of –OH. As a result of C–OH oxidation during the hydrothermal reaction, a C=O peak located at 1631 cm−1 was identified . It was said that the C=O bond, as well as the graphite-liked conjugated structure inside CDs, might be the luminescent donors in CDs. And a C=C stretching vibration peak located at 1594 cm−1 were detected. C=C is also regarded as a source of luminescence for fluorescence.
Compared to pure cellulose CDs, the C–O–C stretching vibration peak located at 1352 cm−1 appeared on the FT-IR spectra of NaOH/urea/cellulose CDs, NaOH/cellulose CDs and urea/cellulose CDs. The reason was as follow: when NaOH or urea was introduced into cellulose system, the ring-opening and oxidation effect of the cellulose glucose structural unit were promoted , and the C–OH were further oxidized by the residual oxygen atoms under the high temperature and pressure environment to yield more C–O–C in CDs . In addition, an extra C–N characteristic absorption peak appeared at 1384 cm−1 in urea/cellulose CDs and NaOH/urea/cellulose CDs, which indicated that N had been successfully doped into CDs in this case.
Elemental composition of the CDs produced from different raw cellulose hydrogels
Proportion of groups of the CDs that produced from different raw cellulose hydrogels
The optimal excitation and emission wavelengths of CDs produced from different raw cellulose hydrogels
Particle size (identified by the molecular weight of the dialysis bag)
Excitation wavelength (nm)
Emission wavelength (nm)
The particle sizes of CDs collected through dialysis bags with different cut-off molecular weights were counted, and the size distribution was plotted and Gaussian fitted (Fig. 4b). As the molecular weight of the dialysis bag decreases, the diameter distribution curves of CDs shift leftward. Corresponding to the “8000D–14000D CDs”, the “3500D–8000D CDs”, the “1000D–3500D CDs” and the “< 1000D CDs”, the mean diameter of CDs are 4.79 ± 1.04 nm, 3.94 ± 1.21 nm, 3.24 ± 0.64 nm and 2.41 ± 0.93 nm, respectively.
3.2 Quantum yield of CDs
Figure 5b revealed the influence of particle size on the QY of CDs that produced from the same raw cellulose hydrogel. The one-way ANOVA analysis result indicated that the particle size will affect the QY of CDs (Sig. = 0.000). The smaller the average particle size is, the higher the QY value is. This is because the smaller quantum dots have a larger proportion of the surface luminescent groups .
3.3 Living cell labeling
For the “< 1000D CDs”, the labeling brightness showed significant difference among the 4 kinds of CDs (Fig. 6b, the Sig. given by one-way ANOVA is 0.000). The labeling brightness of pure cellulose CDs was the lowest (the average gray value was 13.49 ± 2.79). When NaOH was carried in cellulose hydrogel as the raw material, the labeling brightness of so-obtained CDs trends to be improved, but there was no statistical difference (Sig. = 0.216, compared to Cellulose CDs). When urea was applied to the raw cellulose hydrogel, the labeling brightness of so-obtained CDs on Hela cells was significantly increased (Sig. = 0.000, compared to Cellulose CDs), and the NaOH/urea/cellulose CDs had the highest labeling brightness among the 4 kinds of CDs (Sig. = 0.000,compared to Cellulose CDs).
In present study, the QY of CDs increased when NaOH and urea were added into the cellulose hydrogels, and the corresponding photoluminescence intensity increased to yield brighter images. The smaller the particle size of CDs is, the higher the QY is, and the brighter the labeled image is. In addition, the cellular imaging effect of CDs is not only determined by quantum efficiency, but also by the uptake rate of cells. Studies have found that the uptake of nanoparticles by cells is determined by several factors such as the size, shape, specific surface area, surface charge and surface modification of the nanoparticles. It is said that nanoparticles with smaller size are more easily to be taken up by cells, and the doping of nitrogen causes more amino groups on the surface of quantum dots, making quantum dots more readily taken up by cells [31, 32]. Therefore, NaOH/urea/cellulose CDs with the smallest particle size (the “< 1000D CDs”) contributed to the brightest cell labeling image in this work.
The labeling brightness of NaOH/urea/cellulose CDs (the “< 1000D CDs”) is similar to that of ER-tracker (the average gray value of labeled cells was 34.62 ± 8.96 and 34.12 ± 6.66, respectively) on living cells. Compared to TRITC, the labeling brightness of trends to be weaker. The part reason may come from the CDs loss during the Triton treatment and subsequent rinse period of TRITC staining. In addition, the optimal exciting wavelength of CDs produced in this work is 370 nm, however, the exciting wavelength of laser confocal microscope is 405 nm, which leads to a weaker emission than it could be.
3.4 Cytotoxicity of CDs
In present work, cellulose hydrogel was used as the carbon source and the doping-material-carrier to produce CDs and N-doped CDs. The groups in so-obtained CDs were measured by means of UV–Vis absorption spectrum, FT-IR spectrum and XPS to confirm whether the N element has been successfully doped in CDs. The microstructure, the morphology, and the particle size of the CDs were observed by means of HRTEM. The QY value of the CDs was detected to describe its photoluminescent property. The labeling brightness was used as the index to investigate the influence of N-doping, particle size, as well as the cell line on the bioimaging property of the CDs. Finally, the influence of element-doping on the cytotoxicity were measured. The experimental results showed that:
Cellulose hydrogel is a nice carbon source and doping-material-carrier to produce fine N-doped CDs due to its distinct spatial network structure. The urea carried in cellulose hydrogel network achieved to dope N into the CDs, and subsequently improved the QY of the CDs through providing the nitrogen-contained auxochrome group. The fluorescence QY values of 4 kinds of CDs produced in this work decreases in the following sequence: NaOH/urea/cellulose CDs > urea/cellulose CDs > NaOH/cellulose CDs > pure cellulose CDs. For the same kind of CDs, the smaller the particle size is, the higher the QY value is.
The CDs immobilized in the cytoplasm of the living cells, and contributed to a non-specific fluorescent labeling. The labeling brightness is both the QY value of the CDs and the uptake rate of the living cells dependent. For the same cell line, the QY of CDs and the labeling brightness of living cells are significantly linear correlated.
The cytotoxicity CDs is low enough for a long-time observation on living cells.
This work was supported by National Natural Science Foundation of China (21875068).
Compliance with ethical standards
Conflict of interest
The authors have no conflict of interest to declare.
- 7.Gao N, Yang W, Nie H, Gong Y, Jing J, Gao L, Zhang X (2017) Turn-on theranostic fluorescent nanoprobe by electrostatic self-assembly of carbon dots with doxorubicin for targeted cancer cell imaging, in vivo hyaluronidase analysis, and targeted drug delivery. Biosens Bioelectron 96:300–307CrossRefGoogle Scholar