Rapid and Green Fabrication of Carbon Dots for Cellular Imaging and Anti-Counterfeiting Applications.

Carbon dots (CDs) with plenty of favorable properties have been extensively investigated in diverse areas including bioimaging, biomedicine, sensor, energy storage, anti-counterfeiting, photocatalysis, and optoelectronic devices. Herein, a simple, rapid, and green sonochemical-assisted method for fabricating nitrogen-doped CDs has been developed. In this approach, the nitrogen-doped CDs can be obtained through irradiation by intensive ultrasonic waves from ultrasonic probes in 30 min. The achieved CDs exhibit excellent water dispersibility, which can be ascribed to their high functionalization. Importantly, the CDs also demonstrate remarkable fluorescent properties, high photostability, and low cytotoxicity, which can be utilized for multicolor cellular imaging and anti-counterfeiting applications. As far as we know, the sonochemical-assisted method for rapidly synthesizing nitrogen-doped CDs from gelatin has never been reported before. Significantly, the sonochemical-assisted approach to rapidly fabricate CDs is versatile for the facile construction of fluorescent CDs, and the obtained CDs can be potentially used in various areas including bioimaging and anti-counterfeiting.

Introduction

Carbon dots (CDs) have aroused keen interest with their plenty of favorable properties, such as tunable photoluminescence, splendid photostability, high biocompatibility, and electronic transport properties, which made CDs play significant roles in various fields including bioimaging, biomedicine, sensor, energy storage, anti-counterfeiting, photocatalysis, and optoelectronic devices.[1−7] To date, various approaches have emerged to construct CDs, and these methods can be generally sorted as “top-down” and “bottom-up” strategies according to their carbon sources.[5−8] The top-down methods usually would smash bulk carbon sources into smaller units, which were limited by their expensive equipment and high consumption of energy.[9] On the other hand, the bottom-up approaches always employed a more facile and simple manner, such as hydrothermal treatment, pyrolysis, and chemical oxidation.[10−13] In recent years, many new techniques have constantly emerged to construct CDs, which revealed impressive superiorities in their high efficiency and low consumption.[14−19]

Gelatin, a natural polymer, is the product of partial hydrolysis of collagen, which can be divided into photographic gelatin, edible gelatin, and industrial gelatin according to their properties and uses. In recent decades, due to its extensive sources, great biocompatibility, and outstanding biodegradability, gelatin demonstrates excellent performance in the fields of biomaterials, such as drug carriers, artificial skin, and scaffolds for tissue engineering.[23−25] Importantly, gelatin can also be employed to fabricate CDs with all these superiorities. Li and co-workers reported their work on the preparation of luminescent CDs through a hydrothermal process from gelatin.[26] Wang and co-workers synthesized CDs/gelatin composite by a microwave approach.[27] Nevertheless, it is time-consuming to achieve mass production of CDs through hydrothermal treatment, and the CDs/gelatin composite obtained from the microwave method was not suitable for some applications in which only CDs were needed.

In this work, we described the rapid fabrication of nitrogen-doped CDs from gelatin via a sonochemical-assisted green method. Compared with the ultrasound bath, the ultrasound probe demonstrated higher efficiency and effectiveness in the preparation of CDs. As revealed in Figure , with the same ultrasonic frequency and irradiation time, fluorescent CDs can be obtained via the ultrasonic probe strategy, while only some larger polymer nanoparticles were achieved in the ultrasonic bath method. The obtained CDs were prepared from gelatin through simple processing in water without adding any catalyst, acid, or alkali solvents. Moreover, the CDs exhibited excellent water dispersibility, splendid biocompatibility, and remarkable fluorescent properties, which were promising for bioimaging and anti-counterfeiting applications.[28−30] As far as we know, the sonochemical-assisted method for rapidly synthesizing nitrogen-doped CDs from gelatin has never been reported before. Significantly, the sonochemical-assisted approach to rapidly fabricate CDs is versatile for the facile construction of fluorescent CDs from many analogous carbon sources.

Figure 1

Schematic representation showing the rapid fabrication of CDs through a sonochemical-assisted approach with an ultrasound probe and their fluorescence imaging and anti-counterfeiting applications.

Results and Discussion

Rapid Fabrication of Nitrogen-Doped CDs from Gelatin

The CDs were constructed via a green sonochemical approach from gelatin, as illustrated in Figure . Gelatin was hydrolyzed by papain and then irradiated by intensive ultrasonic waves for 30 min using ultrasonic probes. After further routine centrifugation and lyophilization, the nitrogen-doped CDs were achieved. The sonochemical method is rapid, green, and facile, with no harsh conditions or complicated manipulations. Importantly, the obtained nitrogen-doped CDs could demonstrate stable photoluminescence and low cytotoxicity. As a contrast, gelatin was also put in an ultrasonic bath without probes for irradiation at the same time. However, there were few CDs in the product but some larger polymer nanoparticles (PNs). The detailed characterizations would be discussed later.

In this method, it was the cavitation phenomena generated from the intensive ultrasonic waves that can be used to explain the formation of CDs. With the alternative high pressure and low pressure in the liquid medium, many microbubbles would rapidly form, grow, and collapse, which would lead to the formation of special hot spots with high temperatures of over 5000 K, pressures of more than 1000 atm, and heating and cooling rates greater than 1010 Ks–1.[16] It is believed that the condensation and carbonization of gelatin happened during the above acoustic cavitation.[5] Nevertheless, the clear mechanism of CDs formation has not been generally agreed till the present moment. However, it can be inferred from the experiments that, with the same frequency and irradiation time, the carbonization energy for treating gelatin in the ultrasonic bath was not enough to prepare CDs. Excitingly, in our strategy, the CDs could be rapidly prepared via the sonochemical-assisted green method.

Characterization of CDs

To assess the morphology of the achieved nitrogen-doped CDs, high-resolution transmission electron microscopy (HR-TEM) was employed. As revealed in Figure A, the CDs showed a spherical shape and were with a mean diameter of 3.8 nm. Clearly, the inset showed their crystalline fringe, and the regular lattice spacing is 0.18 nm, which remains well with the (102) diffraction planes of sp2 graphitic carbon.[13] These results can prove that the CDs had been successfully prepared via the probe-assisted sonochemical approach. The morphonology of PNs was also investigated, as shown in Figure B. Nevertheless, the PNs were larger than CDs with a mean diameter of 10.2 nm, and no crystalline fringes can be detected in these nanoparticles with irregular shapes. It was reckoned that the condensation of gelation had also taken place during the sonochemical method without probes, but the next carbonization procedure was not finished yet.

Figure 2

(A) HR-TEM image of obtained CDs through an ultrasonic probe approach and the inset showed their crystalline fringe with a regular lattice spacing of 0.18 nm. (B) HR-TEM image of obtained PNs via the ultrasound bath method.

The chemical compositions of the achieved CDs were measured by Fourier transform infrared spectra (FTIR) together with X-ray photoelectron spectra (XPS) analysis. As exhibited in Figure , the characteristic absorption peak at 3293 cm–1 is from O–H/N–H, 1631 cm–1 is for the stretching vibration band of C=O, and 1080 and 874 cm–1 come from the stretching vibration band of C–O, which all indicate that the surface of CDs has been partially oxidized.[30,31] Furthermore, the obvious peak at 1540 cm–1 can be associated with N–H bands of vibration and deformation, which manifested that there are some amino-related groups on the CDs. Moreover, the absorption peaks at 1249 cm–1 can be ascribed to the symmetrical stretching vibration band of C–C, and the anti-symmetrical stretching vibration band of C–C is seated at 1029 cm-1.[32,33] Additionally, the peaks at 2940 and 1405 cm–1 are assigned to the stretching vibrations C–H and C=C, which further suggests the production of alkyl and aryl groups on their surface.[34]Figure A shows the XPS data of CDs. The CDs are primarily composed of carbon (47.03%), oxygen (26.10%), nitrogen (26.72%), and sulfur (0.16%). As revealed in Figure B, the high resolution of the C 1s spectrum displays the peaks at 284.8, 286.0, 286.3, 287.8, and 288.8 eV, which indicates that there are C–C/C=C, C–N, C–O, C=O, and O—C=O groups functionalized on the surface of CDs.[35] The functional groups endow the CDs with great solubility and stability in an aqueous system, which could also be further modified for diverse applications in different areas. Figure C and Figure D show the high resolution of N 1s and O 1s spectra, respectively, which also prove the presence of amino-related functional groups and are consistent with the above FTIR results.

Figure 3

FTIR spectrum of obtained CDs.

Figure 4

(A) XPS survey spectrum of CDs with detailed percent of different elements and high-resolution XPS scanning and their fitting curves of (B) C 1s, (C) N 1s, and (D) O 1s.

The detailed optical qualities of CDs were checked by investigating their UV–vis spectrum and fluorescence (FL) spectra. From Figure A, it can be observed that the CDs reveal a broad absorption band in the range of 300–400 nm, which is attributed to the presence of CDs. The FL spectra in Figure A reveal that the CDs could emit a fluorescence around 450 nm with an excitation wavelength of 365 nm. Clearly, the inset photographs in Figure A show that the aqueous dispersion of CDs is transparent to daylight, whereas it demonstrates intense blue luminescence under UV light (λmax = 365 nm). As depicted in Figure B and Figure C, the CDs featured a typical photoluminescence property that the emission peaks would change with the variation of excitation wavelengths. As the excitation wavelengths changed from 300 to 500 nm, their fluorescence band maximum would shift from 400 to 550 nm. Also, the quantum yield of CDs was measured, and the detailed process can be seen in our earlier research.[36] The result proves that the fluorescent quantum yield of CDs can reach as high as 33.8%. Importantly, the CDs reveal great photostability, as depicted in Figure D, which can be stored for 6 weeks without an obvious decrement of luminescence intensity. These features make the CDs quite favorable for bioimaging applications.

Figure 5

(A) UV–vis spectrum and FL spectrum of CDs; the inset showed the photographs of CDs in daylight and under UV light. (B) FL spectra of CDs with different excitation wavelengths, (C) the normalized FL spectra of CDs with different excitation wavelengths, and (D) the photostability of CDs during several weeks.

Cytotoxicity of CDs in Cells

The cytotoxicity of prepared CDs was investigated with A549 cells via a cell counting kit-8 (CCK-8) assay. As demonstrated in Figure A, the cell viability of A549 cells remained with no obvious changes after different concentrations of CDs were put into the culture media of CDs for 12 and 24 h. Even when the concentration of CDs reached as high as 300 μg/mL, the viabilities of A549 cells remained more than 91% after 24 h. These results proved the excellent biocompatibility of CDs for bioimaging and other bioapplications.

Figure 6

(A) Cell viability of A549 cells toward CDs with diverse concentrations in 12 and 24 h and the fluorescence imaging picture of A549 cells with λex of (B) 408, (C) 532, and (D) 633 nm.

CDs for Celluar Imaging

Taking advantages of their favorable fluorescent properties and excellent biocompatibility, the cell imaging behavior of CDs was detected. The A549 cells were employed as model cells for the experiments. After the cells were incubated with CDs of 50 μg/mL for 3 h, the cells were cleaned and exposed to fluorescence microscopy. As illustrated in Figure B–D, the cytoplasm region of cells could be lightened with intense fluorescence of different colors as the excitation wavelengths changed, which demonstrated that the CDs can be potentially used for multicolor bioimaging of cells. Furthermore, it could be also observed that the morphology of A549 cells still remained well after incubation with CDs for 3 h. These results illustrated that the CDs were promising for bioimaging applications.[37]

CDs for Anti-Counterfeiting

Owing to their great photostability and tunable fluorescent properties, the obtained CDs can be potentially used as an anti-counterfeiting ink. As exhibited in Figure A, the water dispersion of CDs was applied for writing two Chinese characters on the cellulosic papers, which were invisible under white light. After the cellulosic papers were exposed to 532 and 633 nm laser irradiation, the words converted to be visible as bright yellow and red Chinese characters (Figure B and Figure C, respectively). These experiments preliminarily proved the potential of CDs for anti-counterfeiting applications.

Figure 7

(A) Picture of a cellulosic paper in white light with characters written on them using CDs as an anti-counterfeiting ink, (B) picture of the above cellulosic paper under a laser irradiation of 532 nm, and (C) picture of the above cellulosic paper under a laser irradiation of 633 nm.

Conclusions

In one word, we have fabricated a facile and green strategy to rapidly construct CDs from gelatin. In this sonochemical-assisted approach, the nitrogen-doped CDs can be obtained through irradiation by intensive ultrasonic waves from ultrasonic probes in 30 min. As far as we know, this is the first example of the rapid synthesis of nitrogen-doped CDs from gelatin. The achieved CDs exhibit excellent water dispersibility, remarkable fluorescent properties, high photostability, and negligible cytotoxicity. Furthermore, the CDs have been nicely utilized for fluorescence imaging of A549 cells and anti-counterfeiting applications. Significantly, the sonochemical-assisted approach to rapidly fabricate CDs is versatile for the facile construction of fluorescent CDs from many analogous carbon sources, and the obtained CDs can be potentially used in various areas including bioimaging and anti-counterfeiting.

Experimental Section

Materials

Gelatin (type-B, 100 bloom), papain, sodium acetate, and acetic acid were received from Aladdin (Shanghai, China), all of which were analytical reagents. Reagents and consumables for cell experiments were all received from Invitrogen. (California, USA).

Rapid Preparation of CDs

Gelatin (1 g) was dissolved in 200 mL of water at 50 °C under continuous stirring, and then, 30 mg of papain together with several drops of acetic acid buffer (pH 5.0) was put into the above water solution to hydrolyze the gelatin. After stirring for about 30 min, an ultrasonic probe was put below in the obtained solution, which was further irradiated with ultrasound for 30 min at 20 kHz and 720 W (Scientz-2400F, Scientz, China). Then, the above solution was treated with centrifugation for 10 min at 12,000 rpm, and the supernatant containing CDs was separated by a filter membrane of 0.22 μm. The achieved solution of CDs can be stored at 4 °C or dried through further lyophilization. The yield of CDs was calculated to be 41.2%.

Characterization of the CDs

The morphologies and microstructures of CDs were determined by HR-TEM of JEM-2100F. FTIR and XPS of CDs were obtained by a Nicolet IS10 spectrometer (Nicolet, USA) and a Thermo ESCALAB 250Xi spectrometer (Thermo Fisher, USA), respectively. Their optical spectra were checked with a UV-3600 spectrometer (Shimadzu, Japan) and a Fls 1000/FS5 fluorophotometer (Edinburgh, England).

Cytotoxicity of the CDs

The cytotoxicity of CDs can be determined via testing their cell viability, which can be obtained through a CCK-8 assay, and the detailed process can be found in our earlier works.[33,38−42] First, A549 cells were carefully cultured in an incubator at 37 °C with 5% CO2 to ensure their good growth. Before the CCK-8 test, A549 cells were transferred to microplates and cultured for 12 h. Then, CDs in Dulbecco’s modified Eagle’s medium (DMEM) solution were put into each well for further incubation. After careful washing with phosphate buffer saline, the mixture of the CCK-8 dye and culture medium was added to all the above wells and the microplates were put in an incubator for a further 2 h. The optical density was detected at 480 nm with a Flex Station 3 microplate reader (Molecular Devices, USA).

Cellular Imaging

A549 cells were carefully cultured as described above before the imaging experiment, and A549 cells with a density of 1 × 105 cell mL–1 were maintained at 37 °C for 24 h. Then, the A549 cells were transferred into a medium containing CDs of 50 μg/mL for 3 h. After careful washes, the cells were imaged by an FV3000 confocal microscope (Olympus, Japan). For identifying their multicolor luminescence imaging behavior of CDs, different excitation wavelengths (405, 488, and 633 nm) were picked in these imaging experiments.

Anti-Counterfeiting

CDs in water (0.5 mg/mL) were prepared as an anti-counterfeiting ink, which was used for writing on a cellulosic paper. Then, the paper was exposed to lasers of 532 and 633 nm, and their pictures were taken using a camera with the help of filters (590/20 nm single-band bandpass filter and 660/13 nm single-band bandpass filter).