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N, S, I co-doped carbon dots for folic acid and temperature sensing and applied to cellular imaging
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 224 (2020) 117444
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N, S, I co-doped carbon dots for folic acid and temperature sensing and applied to cellular imaging Zhao Mua, Jianhao Huaa, Yaling Yanga* a
Faculty of Life Science and Technology, Kunming University of Science and Technology, Yunnan Province 650500, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 March 2019 Received in revised form 24 July 2019 Accepted 29 July 2019 Available online 30 July 2019
The application of fluorescent carbon dots in bio-imaging has huge positive significance in the field of biomedicine. By taking this advantage, herein we prepared nitrogen, sulfur and iodine doped carbon dots (N,S,I-CDs) by a facile hydrothermal reaction using C3N3S3, potassium iodate (KIO3) and ethylenediamine (EDA), and the obtained N,S,I-CDs show bright blue fluorescence with a high fluorescence quantum yield of about 32.4%. The prepared N, S, I-CDs could interact with the folic acid (FA) with high selectivity, lead to development of a high sensitive method for the FA detection from 0.1 to 175 mM wide linear range with a detection limit of 84 nM (S/N ¼ 3) and also applied them in U-2 OS cells imaging. Moreover, this sensor possessed a good sensitivity, linearity and reversibility in the temperature range of 10e80 C, and successfully applied for the temperature sensing in cell HT-29 samples. This investigation illustrates that as-prepared N, S, I-CDs probe may have great potential as a high-performance platform for the accurate recognition of temperature in cells and could provide a new tool for the detection of FA in cells. © 2019 Elsevier B.V. All rights reserved.
Keywords: Carbon dots Cellular imaging Fluorescence resonance energy transfer Temperature sensing
1. Introduction Folic acid is also known as vitamin B9, which is one of the water-soluble compounds of the vitamin B group and equivalent to pteroylglutamic acid (PGA) [1]. Also, FA plays a vital role in human body for promoting the maturation of young cells in the bone marrow [2]. Yet, the lack of folic acid in humans can cause macrocytic anemia and leukopenia, therefore proper maintenance of FA levels is most important especially for the pregnant women [3e6]. Because, the role FA is to maintain the normal growth and development of fetus by participating in the metabolism of amino acids and nucleic acids in the pregnant women [4,7]. If the body lacks FA in the first trimester, it will not only affect the hematopoietic function, but also causes the anemia during pregnancy [3], leads to fetal growth retardation and obstruction, this situation further cause to fetal neural tube defects [8,9]. In addition, the lack of FA is also associated with cardiovascular disease [10,11], Alzheimer's disease [12,13] and some mental illnesses [14]. However, on the other hand, high levels of folic acid in the human body can lead to zinc deficiency, nausea, vomiting, anorexia and a series of intestinal problems [15]. Hence, the quantitative detection of folic acid has great positive significance. So far, different methods such as high performance liquid chromatography [16], capillary electrophoresis [17], surface-enhanced Raman scattering [18], chemiluminescence [19] and photoelectrochemistry [20e23] have been * Corresponding author. E-mail address: [email protected] (Y. Yanga). https://doi.org/10.1016/j.saa.2019.117444 1386-1425/© 2019 Elsevier B.V. All rights reserved.
applied to FA detection. Among all these methods, fluorescence method has verified as one of the outstanding methods owing to its advantages such as high sensitivity, simplicity in sample preparation, quick response and portability [24]. Among various fluorescence materials carbon quantum dots attained the huge attention because, Carbon quantum dots (CQDs) are a new type of carbon-based zero-dimensional material with extremely small size (below 10 nm) and excellent optical properties [25e29]. Since the first discovery of carbon quantum dots, many synthetic methods have been developed. Also, researchers have been paying more attention because of the wide range of raw materials used for CQDs synthesis are low in cost. The carbon quantum dots synthesized by various methods have many advantages such as good water solubility, low toxicity, environmental friendliness, good biocompatibility, stable optical properties, stable chemical properties, and easy surface functionalization, and are widely used in analysis and detection [26,30], environmental monitoring [31,32], medical imaging [33,34], photocatalysis [35,36] and energy development [37,38], in all these sections CQDS have shown good application prospects. At the same time, the ratiometric fluorescence probes based on the calculation of the ratio of carbon dots at two different emission intensities has been widely used [39]. This method can achieve lower background interference and improve the accuracy and sensitivity of the analytical detection process. Based on the excellent properties of carbon quantum dots, it is practical to develop a fluorescent probe for quantitative detection of folic acid.
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The present investigation demonstrates the fluorescent carbon dots co-doped with nitrogen, sulfur and iodine synthesis by a onestep hydrothermal method. The three-element co-doping enhanced the fluorescence yield and selectivity of the carbon dots. In addition, the N, S, I-CDs show good water solubility, stability, photobleaching resistance and the like. These advantages provided a good basis for quantitative, qualitative analysis and detection potential. The N, S, ICDs were applied for the folic acid detection. A strong hydrogen bond interaction between CQDs and folic acid results in occurrence of fluorescence resonance energy transfer, thereby a ratiometric fluorescence detection was achieved. The N, S, I-CDs also used for temperature sensing, which acts as a fundamental physical parameter and plays an important role in chemical and biological systems. The number of non-radiative channels formed by surface defects of CDs was positively correlated with the temperature, and the relative fluorescence intensity is also linearly related to temperature. Therefore, temperature detection with high sensitivity was achieved. Also, the biocompatible nature of N, S, I-CDS, provided the possibility of imaging cells in vivo. The imaging experiments on U-2 OS and colon cancer cell HT-29 demonstrated that N, S, I-CDs can be applied to folic acid and temperature sensing in vivo and in vitro.
2.2. Instrumentation Fluorescence measurements were carried out on a G9800A Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, USA). The UVevis absorption spectra were recorded by a UV-2600 UVevisible spectrophotometer (SHIMADZU, Japan). The morphological evaluation was performed using a FEI Tecnai G2 F30 transmission electron microscope (TEM). The Fourier transform infrared (FT-IR) spectrum of N/S-CDs was recorded by using a Tensor-27 FTIR spectrometer (Bruker, Germany). Two other instruments of D8advance X-ray diffractometer (XRD) (Bruker, Germany) and KAlphaX photoelectron spectroscopy (XPS) (Thermo Fisher Scientific Inc. U.S.A.) were used for the characterization of N, S, I-CDs. The fluorescence images of cells were observed and photographed by Nikon A1 Laser confocal microscopy. The vortex mixer (Hanuo Instrument Co., Ltd., XH-B, Shanghai, China) and high speed centrifuge (Shanghai Surgical Instrument Factory, 80e2, Shanghai, China) were applied in the whole experiments. 2.3. Synthesis of N, S, I-CDs C3N3S3 powder was prepared from the previously reported literature [40]. Typically, 0.354 g of H3C3N3S3 was added to 100 mL of NaOH aqueous solution (0.06 M) and stirred at 0 C. Then configured the aqueous solution containing 0.761 g of I2 and 2.05 g of KI were dropped slowly into the above solution. The mixture kept stirred for the overnight and at the end a yellow precipitate was observed. The collected yellow precipitate was centrifuged at 8000 rpm for 20 min and washed three times with deionized water, then dried in vacuum at 60 C. N, S, I-CDs were synthesis as follows: C3N3S3 (50 mg), KIO3 (0.5 g) and EDA (3 mL) were mixed with 50 mL of deionized water and sonicated for 10 min to attain the homogeneous suspension by ultrasound. Then the mixture was transferred into a 100 mL Teflonlined stainless-steel autoclave. After heating at 180 C for 8 h, the autoclave was cooled to room temperature. Finally, the aqueous solution was filtered by 0.22 mm membrane to remove larger particles and stored at 4 C (Fig. 1).
2. Experimental section 2.1. Materials Trithiocyanuric acid (C3H3N3S3) was purchased from Beijing Bailingwei Technology Co., Ltd. Iodine (I2), potassium iodide (KI) and potassium iodate (KIO3) were obtained from Shanghai Chemical Reagent General Factory. The analytical reagent of ethylenediamine (EDA) was purchased from Tianjin No.3 Chemical Reagent Plant. The water used for all the experiments were purified through an EasyQ system (LAB-BIOGEN, Kunming, China). Different Amino Acids, CaCl2, ZnCl2, FeCl3$6H2O, dopamine (DA), Uric acid (UA) and Folic acid (FA) were purchased from Aladdin Reagent (Shanghai) Co., Ltd. And all of them were dissolved in ultrapure water (100 mL) to afford 1 mM aqueous solutions, respectively. All samples were prepared at room temperature.
HS
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Folic acid detection N,S,I-CDs 10℃
Temperature detection
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15°C Fig. 1. Schematic illustration of the preparation of N, S, I-CDs, detection of FA and cell imaging of N,S,I-CDs.
N,S,I-CDs
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where F is the fluorescence quantum yield (QY), I is the measured integrated emission intensity, A is the absorbance and h is the refractive index. The subscript S and R were behalf of sample and reference.
2.4. Measurement of fluorescence quantum yield (QY) The fluorescence quantum yield (QY) of N, S, I-CDs was measured according to an established procedure by comparing the fluorescence intensities and absorption values of N, S, I-CDs samples with quinine sulfate. Quinine sulfate was dissolved in 0.1 M H2SO4 (literature quantum yields 0.54 at 360 nm) while N, S, I-CDs were dissolved in water. In order to minimize the reabsorption effects, the absorbance was kept below 0.15. The QY of N, S, I-CDs was computed by the following equation:
.
FS ¼ FR þ ðIS =IR Þ ðAR =AS Þ hS 2 hR 2
3
2.5. N, S, I-CDs for folic acid sensing In a typically assay, 20 mL of N, S, I-CDs stock solution and varying amounts of folic acid solution were added to a 10 mL quartz tube and then dilute to 4 mL with deionized water. After vortexing for 1 min, incubate for 5 min at room temperature. The fluorescence emission spectrum at 388 nm was recorded with a fluorescence spectrophotometer at an excitation wavelength of 318 nm. Under
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Fig. 2. (A) TEM image and The inset shows the size distribution and particle size distribution of N,S,I-CDs. (B) UVeVis absorption, PL excitation and emission spectra of N,S,I-CDs. (C) PL spectra of N,S,I-CDs at different excitation of 288-348 nm. (D) XRD patterns of N,S,I-CDs. (E) FT-IR spectrum of N,S,I-CDs.
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the same conditions, other substances were used instead of folic acid to continue the detection, and the fluorescence emission spectrum data were collected and compared to verify the selectivity of folic acid for N, S, I-CDs (Fig. 1).
2.6. Cell imaging U-2 OS and colon cancer cell HT-29 were mixed with N, S, I-CDs on different 24-well plates and incubated for 2 h. Two hours later, the culture medium containing free N, S, I-CDs were discarded. U-2 OS and colon cancer cells were washed repeatedly with phosphate buffer saline (PBS) for three times each. After processing, the fluorescence images of two different kinds of cells were observed and photographed by Nikon A1 Laser confocal microscopy (Fig. 1).
2.7. Cell toxicity
To observe the morphology of the N, S, I-CDs, transmission electron microscopy (TEM) images were acquired. As described in Fig. 2A, the N, S, I-CDs were appeared in spherical dots and well dispersed with a diameter of 5 nm. The HRTEM image revealed the high crystallinity of the N, S, I-CDs with a lattice spacing of 0.35 nm. Fig. 2B shows the UVevis absorption band, fluorescence excitation and emission spectra of N, S, I-CDs. In UVevis spectrum, the absorption peak at 223 nm may be due to the p-p* transition of C]C bonds. The excitation peak of N, S, I-CDs was observed at 318 nm and emission intensity peak was found at 388 nm. Fig. 2C shows the fluorescence emission intensity at different excitation wavelengths (288e348 nm). The maximum emission intensity was observed at 388 nm with an excitation wavelength of 318 nm. The subsequent experiments were carried out at 318 nm excitation wavelength. As mentioned earlier, the QY of N, S, I-CDs was determined with the help of a reference called quinine sulfate (0.1 M H2SO4 as solvent, QY ¼ 0.54), the observed QY was about 32.4%. Fig. S1 shows that the fluorescence yield of N, S, I-CDs is significantly higher than that of N, S-CDs synthesized without potassium iodate (QY ¼ 26.7%, Fig. S1). The XRD pattern of N, S, I-CDs was shown in Fig. 2D. The observed diffraction pattern centered at 25.0 corresponding to the (002) plane of graphite and this pattern illustrated that the CDs contained the graphite like structure. Furthermore, the structure and surface functional groups of N, S, I-CDs were characterized with FTIR spectra. As shown in Fig. 2E, the broad absorption band at 3392.48 cm1 can be attributed to the stretching vibrations of OeH. The peaks at 3266.46 cm1,
Raw Fit C-C/C=C C-S C-N C=O/C=N C-O
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3.1. Characterization of N, S, I-CDs
I 3d5
U-2 OS and colon cancer cell HT-29 were seeded into two 96well plate in the standard medium at 37 C with 5.0% CO2 atmosphere for 24 h. Then the U-2 OS and colon cancer cells were incubated with different concentrations of N, S, I-CDs for 24 h. Five parallel samples were taken from each group, and the cells without N, S, I-CDs were used as control group. The cell viability was obtained through colorimetric MTT assays (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) to assess every cell. The cells were washed with PBS for three times, then MTT reagent and medium were added to incubate for 4 h, and the cells were lysed with DMSO (dimethyl sulfoxide). Finally, the absorbance of MTT at 570 nm was recorded by microplate reader.
3. Results and discussion
404
162
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Raw Fit S 2p
3/2
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C-S
172
Binding energy (ev)
Fig. 3. (A) XPS survey scan of N,S,I-CDs. (B) C 1s XPS, (C) N 1s XPS and (D) S 2p XPS of N,S,I-CDs.
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2967.16 cm1, 1671.18 cm1 and 1389.07 cm1 were ascribed to the stretching vibrations of NeH, CeH, C]O and CeN, respectively. The absorption band at around 1221.52 cm1 can be assigned to CeS groups. The peak at 1109.82 cm1 corresponds to the CeC bonds. And the stretching vibrations of CeI were located at 527.36 cm1. Based on the above FT-IR data, a large number of hydrophilic groups (hydroxyl, carboxyl) exist on the surface of N, S, I-CDs, which might be the reason for the hydrophilic nature of the synthesized N, S, ICDs. Moreover, the existence of CeI bond further confirmed that the introduction of potassium iodate could be a reason for enhanced fluorescence yield of N, S, I-CDs. X-ray photoelectron spectroscopy (XPS) was used to determine valence states and composition of elements in N, S, I-CDs. The full range XPS spectrum of N, S, I-CDs in Fig. 3A displayed the seven major peaks, for I 4d, S 2p, C 1s, N 1s, O 1s, I 3d5 and I 3d3, respectively. This observation illustrated the existence of C, N, O, S and I in synthesized N, S, I-CDs. In Fig. 3B the C 1s XPS spectrum was deconvoluted into five peaks at 283.1, 283.9, 284.6, 285.8 and
286.4 eV, which were assigned to CeC/C¼C, CeS, CeN, C¼O/C¼N and CeO bonds, respectively. The N 1s XPS spectrum (Fig. 3C) was deconvoluted into three peaks at 396.7, 397.9 and 399.7 eV, which can be assigned to C]N, CeN and OeN bonds, respectively. The groups which were characterized with XPS were in well agreement with the FT-IR result. In addition, the S 2p spectrum (Fig. 3D) had two peaks: the peaks at 166.8 and 167.8 eV assigned to S 2p3/2 and S 2p1/2 respectively. The above characterization discussion illustrated the successful formation of N, S, I-CDs by hydrothermal method. 3.2. Stability and cytotoxicity of N, S, I-CDs In order to explore the stability of N, S, I-CDs, the changes in fluorescence emission intensity were observed at various conditions such as pH, storage time and ionic strength by NaCl. Fig. 4A displayed the normalized values of the fluorescence intensities of N, S, I-CDs at different pH environments, there was no significant change in emission intensity of N, S, I-CDs from pH 2 1.3
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Fig. 4. (A) The normalized fluorescence intensity of N,S,I-CDs at different pH values. (B) The normalized fluorescence intensity of N,S,I-CDs at different storage time. (C) The normalized fluorescence intensity of N,S,I-CDs with different ionic strengths. (D) Viability of U-2 OS after 24 h treatment with N,S,I-CDs as calculated from the MTT assay. (E) Viability of colon cancer cell HT-29 after 24 h treatment with N,S,I-CDs as calculated from the MTT assay.
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to 7. However, at pH ¼ 8, there was a certain fluorescence enhancement, which may be the effect of deprotonation on the surface of N, S, I-CDs. Therefore, N, S, I-CDs have good stability in acidic and neutral pH environments. Fig. 4B shows the effect of storage time on fluorescence intensity. The normalized value of the fluorescence intensity of N, S, I-CDs was not changed significantly with the prolonged storage time, indicating that N, S, ICDs inherited a strong resistant to photobleaching ability. When it comes to ionic strength evaluation (Fig. 4C), the fluorescence intensity of N, S, I-CDs did not change significantly in aqueous solutions even at different ionic strengths. In summary, N, S, ICDs acquired a good stability in many aspects, which is beneficial to the detection experiments and cell imaging of ions and small molecules. In order to explore the compatibility of N, S, I-CDs to cell imaging, colorimetric MTT assays were used to verify the cytotoxicity of N, S, I-CDs at different concentrations. The specific data was shown in Fig. 4D and E. With increasing of N, S, I-CDs, the gradual decline in cell viability was observed. However, in the presence of high concentrations of N, S, I-CDs, cell viability was as high as 86% and 87%, demonstrating that N, S, I-CDs have good biocompatibility and low cytotoxicity. 3.3. Response of N, S, I-CDs to FA and cell imaging Because of their excellent optical properties, N, S, I-CDs can be used as fluorescent probes for qualitative analysis and quantitative detection of substances with good selectivity. Fig. 5(A) displayed the interference result of N, S, I-CDs. The changes in fluorescence intensity of N, S, I-CDs were measured after adding a series of substances (Fig. S2). There were no substantial changes that were observed initially, however, after the addition of FA, the emission
intensity was quenched significantly and at the same time a new emission peak was observed at 458 nm. Under the optimum conditions (25 C, pH ¼ 7, excitation wavelength of 318 nm and kept for 5 min, Fig. S3), N, S, I-CDs were used as a fluorescent probe to detect the folic acid. Fig. 5(B) showed the quantitative determination of folic acid in the presence of N, S, I-CDs. The fluorescence emission intensity of N, S, I-CDs changed gradually with the increasing concentration of folic acid from 0 to 175 mM. There was a continuous decline in intensity of N, S, I-CDs at 388 nm but at the same time a new emission peak was developed at 458 nm simultaneously, interestingly, this new emission peak intensity was raising with increasing concentration of FA and decreasing emission intensity of N, S, I-CDs at 388 nm. We could see the enhancing emission intensity at 458 nm fluorescence emission peak and declining emission intensity at 388 nm. Finally, the fluorescence emission peak at 388 nm was almost disappeared and a new fluorescence emission peak at 458 nm was completely displayed. Therefore, the concentration of folic acid can be quantitatively analyzed by the ratio of fluorescence intensity at 388 and 458 nm (I458/I388). The I458/I388 ratio increased proportionally with the increase of folic acid concentration, showing a good linear relationship. The linear regression equation being I458/ I388 ¼ 0.012CFA þ 0.1747 (R2 ¼ 0.996, Fig. 5C), and the detection limit of FA is 84 nM. The detection range was from 0.1 to 175 mM. This experiment proved that N, S, I-CDs have good selectivity, high sensitivity and wide linear range in the detection of folic acid. N, S, ICDs are a new fluorescent probe for the folic acid detection. The mechanism behind the detection of FA by N, S, I-CDs may be due to the fluorescence resonance energy transfer. From the above characterization data, we could confirm that there was many carboxyl, hydroxyl groups and iodide ions on the surface of the N, S, I-CDs. At the same time, folic acid structure contains amino and carboxyl groups. When the N, S, I-CDs are in contact with folic acid, 600
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Fig. 5. (A) Fluorescence intensity variation of N,S,I-CDs in the presence of different substances. (B) Fluorescence intensity variation of N,S,I-CDs after adding different concentrations of FA (0e175 mM). (C) Linear relationship between I458/I388 and the concentration of FA. (D) UVeVis absorption of N,S,I-CDs, FA and N,S,I-CDs þ FA, and PL emission spectra of N,S,ICDs.
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strong hydrogen bonding interactions occur rapidly and pull closer. Further, as can be seen from Fig. 5D, the fluorescence emission peak of the N, S, I-CDs partially overlapped with the UVeVis absorption peak of folic acid. This result could explain the possibility of fluorescence resonance energy transfer. With increasing concentration of folic acid, a part of the energy of the N, S, I-CDs might transferred to the folic acid, leads to decrease in the energy of the N, S, I-CDs due to the strong hydrogen bond interaction, proving that the intensity of the fluorescence emission peak was weakened at 388 nm. A new fluorescence emission peak appeared at 458 nm after the energy of the folic acid was increased. According to the previous report [38], the shifts in the fluorescence emission wavelengths from both the N, S, I-CDs and folic acid were due to the delocalization of p electrons of folic acid in the N, S, I-CDs/folic acid system. Aiming at the phenomenon of folic acid induced fluorescence resonance energy transfer of N, S, I-CDs, a scheme of detecting folic acid in living cells by N, S, I-CDs was proposed. Two different concentrations of folic acid (80 mM and 175 mM) were added to the pretreated U-2OS and incubated in the growth medium for 20 min at 37 C. It can be seen from Fig. 6 that the blue fluorescence was weakened, and the corresponding green fluorescence was enhanced after folic acid was added to the blank group. The overall fluorescence color changed from blue fluorescence of Fig. 6A4 to green fluorescence of Fig. 6C4. Blue fluorescence corresponds to the fluorescence intensity at 388 nm, while green fluorescence corresponds to 458 nm. Therefore, the change of cell fluorescence color after adding folic acid was consistent with the fluorescence spectrum of N, S, I-CDs in presence of folic acid. Therefore, N, S, I-CDs can be used as fluorescent probes to detect folic acid in cells.
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3.4. Response of N, S, I-CDs to temperature and cell imaging The effect of temperature on the fluorescence intensity of N, S, ICDs was analyzed in the process of studying and experimenting on the properties of N, S, I-CDs. It was found that the fluorescence intensity of N, S, I-CDs was proportional to the temperature in the range of 10e80 C (Fig. 7A). The fluorescence intensity had absolute dependence on the temperature in the process of varying the temperature. Fig. 7B showed the linear regression equation between I/I0 and temperature: I/I0 ¼ -0.01 t þ 1.0975 (R2 ¼ 0.9947). Obviously, I/I0 decreased by 68% when the temperature was raised from 10 C to 80 C. Also, it exhibited the accuracy of temperature measurement by N, S, I-CDs was high. The stability of temperature response towards N, S, I-CDs was tested by cyclic experiments. Temperature was changed continuously in the range of 10e80 C. After five cycles, as shown by Fig. 7C, it still maintained a good dependence. It showed that N, S, I-CDs gained good stability for temperature detection. The mechanism behind the response of N, S, I-CDs to temperature may be due to the increase in number of nonradiative channels on surface defects with raise in temperature. The increase in the number of non-radiative channels directly results in more electrons returning from the excited state to the ground state, thus weakening the fluorescence intensity. On the contrary, as the temperature decreases, the number of non-radiative channels decreases, and the electrons return to the excited state to restore the fluorescence intensity. Therefore, N, S, I-CDs are stable temperature detectors. Based on the above experimental results, the N, S, I-CDs can be a good fluorescent probe for biological imaging of cells too.
Fig. 6. Confocal microscopy images of U-2 OS incubated with 200 mg mL1 N,S,I-CDs (A1-A4) and after addition of 80 mM (B1-B4) and 175 mM (C1-C4) of FA in the culture solution (measured temperature: 25 C).
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Fig. 7. (A) Fluorescence intensity variation of N,S,I-CDs at different temperatures. (B) I/I0-temperature plots of N,S,I-CDs during 10e80 C. (C) The cycle experiment of the temperature response with N,S,I-CDs.
Therefore, based on the characteristics of N, S, I-CDs as a temperature sensor, we studied the temperature dependence of N, S, I-CDs in colon cancer cell HT-29. The confocal laser scanning microscopy image in Fig. 8 displayed the cell imaging of N, S, I-CDs and it exhibited a good cell morphology. We can see that the N, S, I-CDslabeled colon cancer cell HT-29 displayed an image with bright blue color spots at 15 C (Fig. 8A). As the temperature increases, the blue color spots were decreased in number at 25 C (Fig. 8B), and on further increase in temperature the blue color becomes significantly darker at 35 C (Fig. 8C). When the temperature was lowered to 15 C (Fig. 8D), the N, S, I-CDs-labeled colon cancer cell HT-29 returned to a bright blue color. More importantly, the fluorescence intensity of the labeled cells did not significantly decreased after 20 min of continuous excitation. The observed result confirms that N, S, I-CDs can be used in biomedical applications while performing the cell labelling and can be used in industries for temperature detection.
4. Conclusions In summary, N, S, I-CDs with high fluorescence quantum yield (32.4%) were successfully synthesized by one-step hydrothermal method using C3N3S3 and EDA as precursors, potassium iodate as additives to improve the fluorescence yield. A series of experiments were proved that N, S, I-CDs have good stability, hydrophilicity, low toxicity and good biocompatibility. This N, S, I-CDs displayed a significant response to the folic acid detection in the linear range of 0.1e175 mM with a promising detection limit of 84 nM. It had shown the stable temperature dependence emission in the range of 10e80 C, at the same time, stable reversibility of emission also seen. In addition, due to the less toxicity of N, S, I-CDs, they were successfully utilized as a nice cellular imaging agent in U-2 OS and colon cancer cell HT-29. Therefore, the synthesized N, S, I-CDs have a broad development in the field of biomedicine and environmental application after further development.
Fig. 8. Confocal microscopy images of N,S,I-CDs-colon cancer cell HT-29 with corresponding fluorescence field at 15, 25, 35 and 15 C, respectively.
Z. Mua et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 224 (2020) 117444
Acknowledgment This study was supported by the Analysis and Testing Foundation of Kunming University of Science and Technology (2018M20172118065), Yunnan Province, China. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.saa.2019.117444.
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[20]
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N, S, I co-doped carbon dots for folic acid and temperature sensing and applied to cellular imaging Zhao Mua, Jianhao Huaa, Yaling Yanga* a
Faculty of Life Science and Technology, Kunming University of Science and Technology, Yunnan Province 650500, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 March 2019 Received in revised form 24 July 2019 Accepted 29 July 2019 Available online 30 July 2019
The application of fluorescent carbon dots in bio-imaging has huge positive significance in the field of biomedicine. By taking this advantage, herein we prepared nitrogen, sulfur and iodine doped carbon dots (N,S,I-CDs) by a facile hydrothermal reaction using C3N3S3, potassium iodate (KIO3) and ethylenediamine (EDA), and the obtained N,S,I-CDs show bright blue fluorescence with a high fluorescence quantum yield of about 32.4%. The prepared N, S, I-CDs could interact with the folic acid (FA) with high selectivity, lead to development of a high sensitive method for the FA detection from 0.1 to 175 mM wide linear range with a detection limit of 84 nM (S/N ¼ 3) and also applied them in U-2 OS cells imaging. Moreover, this sensor possessed a good sensitivity, linearity and reversibility in the temperature range of 10e80 C, and successfully applied for the temperature sensing in cell HT-29 samples. This investigation illustrates that as-prepared N, S, I-CDs probe may have great potential as a high-performance platform for the accurate recognition of temperature in cells and could provide a new tool for the detection of FA in cells. © 2019 Elsevier B.V. All rights reserved.
Keywords: Carbon dots Cellular imaging Fluorescence resonance energy transfer Temperature sensing
1. Introduction Folic acid is also known as vitamin B9, which is one of the water-soluble compounds of the vitamin B group and equivalent to pteroylglutamic acid (PGA) [1]. Also, FA plays a vital role in human body for promoting the maturation of young cells in the bone marrow [2]. Yet, the lack of folic acid in humans can cause macrocytic anemia and leukopenia, therefore proper maintenance of FA levels is most important especially for the pregnant women [3e6]. Because, the role FA is to maintain the normal growth and development of fetus by participating in the metabolism of amino acids and nucleic acids in the pregnant women [4,7]. If the body lacks FA in the first trimester, it will not only affect the hematopoietic function, but also causes the anemia during pregnancy [3], leads to fetal growth retardation and obstruction, this situation further cause to fetal neural tube defects [8,9]. In addition, the lack of FA is also associated with cardiovascular disease [10,11], Alzheimer's disease [12,13] and some mental illnesses [14]. However, on the other hand, high levels of folic acid in the human body can lead to zinc deficiency, nausea, vomiting, anorexia and a series of intestinal problems [15]. Hence, the quantitative detection of folic acid has great positive significance. So far, different methods such as high performance liquid chromatography [16], capillary electrophoresis [17], surface-enhanced Raman scattering [18], chemiluminescence [19] and photoelectrochemistry [20e23] have been * Corresponding author. E-mail address: [email protected] (Y. Yanga). https://doi.org/10.1016/j.saa.2019.117444 1386-1425/© 2019 Elsevier B.V. All rights reserved.
applied to FA detection. Among all these methods, fluorescence method has verified as one of the outstanding methods owing to its advantages such as high sensitivity, simplicity in sample preparation, quick response and portability [24]. Among various fluorescence materials carbon quantum dots attained the huge attention because, Carbon quantum dots (CQDs) are a new type of carbon-based zero-dimensional material with extremely small size (below 10 nm) and excellent optical properties [25e29]. Since the first discovery of carbon quantum dots, many synthetic methods have been developed. Also, researchers have been paying more attention because of the wide range of raw materials used for CQDs synthesis are low in cost. The carbon quantum dots synthesized by various methods have many advantages such as good water solubility, low toxicity, environmental friendliness, good biocompatibility, stable optical properties, stable chemical properties, and easy surface functionalization, and are widely used in analysis and detection [26,30], environmental monitoring [31,32], medical imaging [33,34], photocatalysis [35,36] and energy development [37,38], in all these sections CQDS have shown good application prospects. At the same time, the ratiometric fluorescence probes based on the calculation of the ratio of carbon dots at two different emission intensities has been widely used [39]. This method can achieve lower background interference and improve the accuracy and sensitivity of the analytical detection process. Based on the excellent properties of carbon quantum dots, it is practical to develop a fluorescent probe for quantitative detection of folic acid.
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The present investigation demonstrates the fluorescent carbon dots co-doped with nitrogen, sulfur and iodine synthesis by a onestep hydrothermal method. The three-element co-doping enhanced the fluorescence yield and selectivity of the carbon dots. In addition, the N, S, I-CDs show good water solubility, stability, photobleaching resistance and the like. These advantages provided a good basis for quantitative, qualitative analysis and detection potential. The N, S, ICDs were applied for the folic acid detection. A strong hydrogen bond interaction between CQDs and folic acid results in occurrence of fluorescence resonance energy transfer, thereby a ratiometric fluorescence detection was achieved. The N, S, I-CDs also used for temperature sensing, which acts as a fundamental physical parameter and plays an important role in chemical and biological systems. The number of non-radiative channels formed by surface defects of CDs was positively correlated with the temperature, and the relative fluorescence intensity is also linearly related to temperature. Therefore, temperature detection with high sensitivity was achieved. Also, the biocompatible nature of N, S, I-CDS, provided the possibility of imaging cells in vivo. The imaging experiments on U-2 OS and colon cancer cell HT-29 demonstrated that N, S, I-CDs can be applied to folic acid and temperature sensing in vivo and in vitro.
2.2. Instrumentation Fluorescence measurements were carried out on a G9800A Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, USA). The UVevis absorption spectra were recorded by a UV-2600 UVevisible spectrophotometer (SHIMADZU, Japan). The morphological evaluation was performed using a FEI Tecnai G2 F30 transmission electron microscope (TEM). The Fourier transform infrared (FT-IR) spectrum of N/S-CDs was recorded by using a Tensor-27 FTIR spectrometer (Bruker, Germany). Two other instruments of D8advance X-ray diffractometer (XRD) (Bruker, Germany) and KAlphaX photoelectron spectroscopy (XPS) (Thermo Fisher Scientific Inc. U.S.A.) were used for the characterization of N, S, I-CDs. The fluorescence images of cells were observed and photographed by Nikon A1 Laser confocal microscopy. The vortex mixer (Hanuo Instrument Co., Ltd., XH-B, Shanghai, China) and high speed centrifuge (Shanghai Surgical Instrument Factory, 80e2, Shanghai, China) were applied in the whole experiments. 2.3. Synthesis of N, S, I-CDs C3N3S3 powder was prepared from the previously reported literature [40]. Typically, 0.354 g of H3C3N3S3 was added to 100 mL of NaOH aqueous solution (0.06 M) and stirred at 0 C. Then configured the aqueous solution containing 0.761 g of I2 and 2.05 g of KI were dropped slowly into the above solution. The mixture kept stirred for the overnight and at the end a yellow precipitate was observed. The collected yellow precipitate was centrifuged at 8000 rpm for 20 min and washed three times with deionized water, then dried in vacuum at 60 C. N, S, I-CDs were synthesis as follows: C3N3S3 (50 mg), KIO3 (0.5 g) and EDA (3 mL) were mixed with 50 mL of deionized water and sonicated for 10 min to attain the homogeneous suspension by ultrasound. Then the mixture was transferred into a 100 mL Teflonlined stainless-steel autoclave. After heating at 180 C for 8 h, the autoclave was cooled to room temperature. Finally, the aqueous solution was filtered by 0.22 mm membrane to remove larger particles and stored at 4 C (Fig. 1).
2. Experimental section 2.1. Materials Trithiocyanuric acid (C3H3N3S3) was purchased from Beijing Bailingwei Technology Co., Ltd. Iodine (I2), potassium iodide (KI) and potassium iodate (KIO3) were obtained from Shanghai Chemical Reagent General Factory. The analytical reagent of ethylenediamine (EDA) was purchased from Tianjin No.3 Chemical Reagent Plant. The water used for all the experiments were purified through an EasyQ system (LAB-BIOGEN, Kunming, China). Different Amino Acids, CaCl2, ZnCl2, FeCl3$6H2O, dopamine (DA), Uric acid (UA) and Folic acid (FA) were purchased from Aladdin Reagent (Shanghai) Co., Ltd. And all of them were dissolved in ultrapure water (100 mL) to afford 1 mM aqueous solutions, respectively. All samples were prepared at room temperature.
HS
H
N
S
Iodine N
N
polymerization
H S
N
SH
N N HS
N N
S S N
S S
N N
SH
NH 2
H 2N
KIO3
N S
S
180°C 8h
S N N
HS
H
N SH
N,S,I-CDs N,S,I-CDs+Folic acid
Folic acid
N,S,I-CDs+Folic acid
Folic acid detection N,S,I-CDs 10℃
Temperature detection
80℃
35°C
15°C Fig. 1. Schematic illustration of the preparation of N, S, I-CDs, detection of FA and cell imaging of N,S,I-CDs.
N,S,I-CDs
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where F is the fluorescence quantum yield (QY), I is the measured integrated emission intensity, A is the absorbance and h is the refractive index. The subscript S and R were behalf of sample and reference.
2.4. Measurement of fluorescence quantum yield (QY) The fluorescence quantum yield (QY) of N, S, I-CDs was measured according to an established procedure by comparing the fluorescence intensities and absorption values of N, S, I-CDs samples with quinine sulfate. Quinine sulfate was dissolved in 0.1 M H2SO4 (literature quantum yields 0.54 at 360 nm) while N, S, I-CDs were dissolved in water. In order to minimize the reabsorption effects, the absorbance was kept below 0.15. The QY of N, S, I-CDs was computed by the following equation:
.
FS ¼ FR þ ðIS =IR Þ ðAR =AS Þ hS 2 hR 2
3
2.5. N, S, I-CDs for folic acid sensing In a typically assay, 20 mL of N, S, I-CDs stock solution and varying amounts of folic acid solution were added to a 10 mL quartz tube and then dilute to 4 mL with deionized water. After vortexing for 1 min, incubate for 5 min at room temperature. The fluorescence emission spectrum at 388 nm was recorded with a fluorescence spectrophotometer at an excitation wavelength of 318 nm. Under
B
Em
800
Absorbance
40 35 30
Distribution (%)
Ex
25 20
600 400 200
15
FL Intensit y( a.u. )
1000
10 5
0
0 1-2
2-3
3-4
4-5
5-6
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7-8
Size (nm)
200
250
300
350
400
450
500
550
600
Wavelength (nm) 800
FL Intensity(a.u.)
700
D
C 288nm 298nm 308nm 318nm 328nm 338nm 348nm
600 500 400 300 200 100 0 350
400
450
500
550
600
Wavelength (nm)
20
30
40
50
60
70
80
2 Theta (degree)
C-I
Transmittance (%)
E C-O C-S C-N
C-H C=O
N-H O-H
4000 3500 3000 2500 2000 1500 1000 500
Wavenumber (cm
-1
)
Fig. 2. (A) TEM image and The inset shows the size distribution and particle size distribution of N,S,I-CDs. (B) UVeVis absorption, PL excitation and emission spectra of N,S,I-CDs. (C) PL spectra of N,S,I-CDs at different excitation of 288-348 nm. (D) XRD patterns of N,S,I-CDs. (E) FT-IR spectrum of N,S,I-CDs.
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the same conditions, other substances were used instead of folic acid to continue the detection, and the fluorescence emission spectrum data were collected and compared to verify the selectivity of folic acid for N, S, I-CDs (Fig. 1).
2.6. Cell imaging U-2 OS and colon cancer cell HT-29 were mixed with N, S, I-CDs on different 24-well plates and incubated for 2 h. Two hours later, the culture medium containing free N, S, I-CDs were discarded. U-2 OS and colon cancer cells were washed repeatedly with phosphate buffer saline (PBS) for three times each. After processing, the fluorescence images of two different kinds of cells were observed and photographed by Nikon A1 Laser confocal microscopy (Fig. 1).
2.7. Cell toxicity
To observe the morphology of the N, S, I-CDs, transmission electron microscopy (TEM) images were acquired. As described in Fig. 2A, the N, S, I-CDs were appeared in spherical dots and well dispersed with a diameter of 5 nm. The HRTEM image revealed the high crystallinity of the N, S, I-CDs with a lattice spacing of 0.35 nm. Fig. 2B shows the UVevis absorption band, fluorescence excitation and emission spectra of N, S, I-CDs. In UVevis spectrum, the absorption peak at 223 nm may be due to the p-p* transition of C]C bonds. The excitation peak of N, S, I-CDs was observed at 318 nm and emission intensity peak was found at 388 nm. Fig. 2C shows the fluorescence emission intensity at different excitation wavelengths (288e348 nm). The maximum emission intensity was observed at 388 nm with an excitation wavelength of 318 nm. The subsequent experiments were carried out at 318 nm excitation wavelength. As mentioned earlier, the QY of N, S, I-CDs was determined with the help of a reference called quinine sulfate (0.1 M H2SO4 as solvent, QY ¼ 0.54), the observed QY was about 32.4%. Fig. S1 shows that the fluorescence yield of N, S, I-CDs is significantly higher than that of N, S-CDs synthesized without potassium iodate (QY ¼ 26.7%, Fig. S1). The XRD pattern of N, S, I-CDs was shown in Fig. 2D. The observed diffraction pattern centered at 25.0 corresponding to the (002) plane of graphite and this pattern illustrated that the CDs contained the graphite like structure. Furthermore, the structure and surface functional groups of N, S, I-CDs were characterized with FTIR spectra. As shown in Fig. 2E, the broad absorption band at 3392.48 cm1 can be attributed to the stretching vibrations of OeH. The peaks at 3266.46 cm1,
Raw Fit C-C/C=C C-S C-N C=O/C=N C-O
Intensity (a.u.)
I 3d3 O 1s
N 1s
C 1s
200
400
600
800
280
282
Binding energy (ev)
400
288
402
S 2p
Intensity (a.u.)
Intensity (a.u.)
398
Binding energy (ev)
286
D
Raw Fit C=N C-N O-N
396
284
Binding energy (ev) N 1s
C
394
C 1s
B
S 2p
I 4d
Intensity (a.u.)
A
0
3.1. Characterization of N, S, I-CDs
I 3d5
U-2 OS and colon cancer cell HT-29 were seeded into two 96well plate in the standard medium at 37 C with 5.0% CO2 atmosphere for 24 h. Then the U-2 OS and colon cancer cells were incubated with different concentrations of N, S, I-CDs for 24 h. Five parallel samples were taken from each group, and the cells without N, S, I-CDs were used as control group. The cell viability was obtained through colorimetric MTT assays (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) to assess every cell. The cells were washed with PBS for three times, then MTT reagent and medium were added to incubate for 4 h, and the cells were lysed with DMSO (dimethyl sulfoxide). Finally, the absorbance of MTT at 570 nm was recorded by microplate reader.
3. Results and discussion
404
162
164
166
168
170
Raw Fit S 2p
3/2
C-S
S 2p
1/2
C-S
172
Binding energy (ev)
Fig. 3. (A) XPS survey scan of N,S,I-CDs. (B) C 1s XPS, (C) N 1s XPS and (D) S 2p XPS of N,S,I-CDs.
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2967.16 cm1, 1671.18 cm1 and 1389.07 cm1 were ascribed to the stretching vibrations of NeH, CeH, C]O and CeN, respectively. The absorption band at around 1221.52 cm1 can be assigned to CeS groups. The peak at 1109.82 cm1 corresponds to the CeC bonds. And the stretching vibrations of CeI were located at 527.36 cm1. Based on the above FT-IR data, a large number of hydrophilic groups (hydroxyl, carboxyl) exist on the surface of N, S, I-CDs, which might be the reason for the hydrophilic nature of the synthesized N, S, ICDs. Moreover, the existence of CeI bond further confirmed that the introduction of potassium iodate could be a reason for enhanced fluorescence yield of N, S, I-CDs. X-ray photoelectron spectroscopy (XPS) was used to determine valence states and composition of elements in N, S, I-CDs. The full range XPS spectrum of N, S, I-CDs in Fig. 3A displayed the seven major peaks, for I 4d, S 2p, C 1s, N 1s, O 1s, I 3d5 and I 3d3, respectively. This observation illustrated the existence of C, N, O, S and I in synthesized N, S, I-CDs. In Fig. 3B the C 1s XPS spectrum was deconvoluted into five peaks at 283.1, 283.9, 284.6, 285.8 and
286.4 eV, which were assigned to CeC/C¼C, CeS, CeN, C¼O/C¼N and CeO bonds, respectively. The N 1s XPS spectrum (Fig. 3C) was deconvoluted into three peaks at 396.7, 397.9 and 399.7 eV, which can be assigned to C]N, CeN and OeN bonds, respectively. The groups which were characterized with XPS were in well agreement with the FT-IR result. In addition, the S 2p spectrum (Fig. 3D) had two peaks: the peaks at 166.8 and 167.8 eV assigned to S 2p3/2 and S 2p1/2 respectively. The above characterization discussion illustrated the successful formation of N, S, I-CDs by hydrothermal method. 3.2. Stability and cytotoxicity of N, S, I-CDs In order to explore the stability of N, S, I-CDs, the changes in fluorescence emission intensity were observed at various conditions such as pH, storage time and ionic strength by NaCl. Fig. 4A displayed the normalized values of the fluorescence intensities of N, S, I-CDs at different pH environments, there was no significant change in emission intensity of N, S, I-CDs from pH 2 1.3
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Fig. 4. (A) The normalized fluorescence intensity of N,S,I-CDs at different pH values. (B) The normalized fluorescence intensity of N,S,I-CDs at different storage time. (C) The normalized fluorescence intensity of N,S,I-CDs with different ionic strengths. (D) Viability of U-2 OS after 24 h treatment with N,S,I-CDs as calculated from the MTT assay. (E) Viability of colon cancer cell HT-29 after 24 h treatment with N,S,I-CDs as calculated from the MTT assay.
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to 7. However, at pH ¼ 8, there was a certain fluorescence enhancement, which may be the effect of deprotonation on the surface of N, S, I-CDs. Therefore, N, S, I-CDs have good stability in acidic and neutral pH environments. Fig. 4B shows the effect of storage time on fluorescence intensity. The normalized value of the fluorescence intensity of N, S, I-CDs was not changed significantly with the prolonged storage time, indicating that N, S, ICDs inherited a strong resistant to photobleaching ability. When it comes to ionic strength evaluation (Fig. 4C), the fluorescence intensity of N, S, I-CDs did not change significantly in aqueous solutions even at different ionic strengths. In summary, N, S, ICDs acquired a good stability in many aspects, which is beneficial to the detection experiments and cell imaging of ions and small molecules. In order to explore the compatibility of N, S, I-CDs to cell imaging, colorimetric MTT assays were used to verify the cytotoxicity of N, S, I-CDs at different concentrations. The specific data was shown in Fig. 4D and E. With increasing of N, S, I-CDs, the gradual decline in cell viability was observed. However, in the presence of high concentrations of N, S, I-CDs, cell viability was as high as 86% and 87%, demonstrating that N, S, I-CDs have good biocompatibility and low cytotoxicity. 3.3. Response of N, S, I-CDs to FA and cell imaging Because of their excellent optical properties, N, S, I-CDs can be used as fluorescent probes for qualitative analysis and quantitative detection of substances with good selectivity. Fig. 5(A) displayed the interference result of N, S, I-CDs. The changes in fluorescence intensity of N, S, I-CDs were measured after adding a series of substances (Fig. S2). There were no substantial changes that were observed initially, however, after the addition of FA, the emission
intensity was quenched significantly and at the same time a new emission peak was observed at 458 nm. Under the optimum conditions (25 C, pH ¼ 7, excitation wavelength of 318 nm and kept for 5 min, Fig. S3), N, S, I-CDs were used as a fluorescent probe to detect the folic acid. Fig. 5(B) showed the quantitative determination of folic acid in the presence of N, S, I-CDs. The fluorescence emission intensity of N, S, I-CDs changed gradually with the increasing concentration of folic acid from 0 to 175 mM. There was a continuous decline in intensity of N, S, I-CDs at 388 nm but at the same time a new emission peak was developed at 458 nm simultaneously, interestingly, this new emission peak intensity was raising with increasing concentration of FA and decreasing emission intensity of N, S, I-CDs at 388 nm. We could see the enhancing emission intensity at 458 nm fluorescence emission peak and declining emission intensity at 388 nm. Finally, the fluorescence emission peak at 388 nm was almost disappeared and a new fluorescence emission peak at 458 nm was completely displayed. Therefore, the concentration of folic acid can be quantitatively analyzed by the ratio of fluorescence intensity at 388 and 458 nm (I458/I388). The I458/I388 ratio increased proportionally with the increase of folic acid concentration, showing a good linear relationship. The linear regression equation being I458/ I388 ¼ 0.012CFA þ 0.1747 (R2 ¼ 0.996, Fig. 5C), and the detection limit of FA is 84 nM. The detection range was from 0.1 to 175 mM. This experiment proved that N, S, I-CDs have good selectivity, high sensitivity and wide linear range in the detection of folic acid. N, S, ICDs are a new fluorescent probe for the folic acid detection. The mechanism behind the detection of FA by N, S, I-CDs may be due to the fluorescence resonance energy transfer. From the above characterization data, we could confirm that there was many carboxyl, hydroxyl groups and iodide ions on the surface of the N, S, I-CDs. At the same time, folic acid structure contains amino and carboxyl groups. When the N, S, I-CDs are in contact with folic acid, 600
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Fig. 5. (A) Fluorescence intensity variation of N,S,I-CDs in the presence of different substances. (B) Fluorescence intensity variation of N,S,I-CDs after adding different concentrations of FA (0e175 mM). (C) Linear relationship between I458/I388 and the concentration of FA. (D) UVeVis absorption of N,S,I-CDs, FA and N,S,I-CDs þ FA, and PL emission spectra of N,S,ICDs.
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strong hydrogen bonding interactions occur rapidly and pull closer. Further, as can be seen from Fig. 5D, the fluorescence emission peak of the N, S, I-CDs partially overlapped with the UVeVis absorption peak of folic acid. This result could explain the possibility of fluorescence resonance energy transfer. With increasing concentration of folic acid, a part of the energy of the N, S, I-CDs might transferred to the folic acid, leads to decrease in the energy of the N, S, I-CDs due to the strong hydrogen bond interaction, proving that the intensity of the fluorescence emission peak was weakened at 388 nm. A new fluorescence emission peak appeared at 458 nm after the energy of the folic acid was increased. According to the previous report [38], the shifts in the fluorescence emission wavelengths from both the N, S, I-CDs and folic acid were due to the delocalization of p electrons of folic acid in the N, S, I-CDs/folic acid system. Aiming at the phenomenon of folic acid induced fluorescence resonance energy transfer of N, S, I-CDs, a scheme of detecting folic acid in living cells by N, S, I-CDs was proposed. Two different concentrations of folic acid (80 mM and 175 mM) were added to the pretreated U-2OS and incubated in the growth medium for 20 min at 37 C. It can be seen from Fig. 6 that the blue fluorescence was weakened, and the corresponding green fluorescence was enhanced after folic acid was added to the blank group. The overall fluorescence color changed from blue fluorescence of Fig. 6A4 to green fluorescence of Fig. 6C4. Blue fluorescence corresponds to the fluorescence intensity at 388 nm, while green fluorescence corresponds to 458 nm. Therefore, the change of cell fluorescence color after adding folic acid was consistent with the fluorescence spectrum of N, S, I-CDs in presence of folic acid. Therefore, N, S, I-CDs can be used as fluorescent probes to detect folic acid in cells.
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3.4. Response of N, S, I-CDs to temperature and cell imaging The effect of temperature on the fluorescence intensity of N, S, ICDs was analyzed in the process of studying and experimenting on the properties of N, S, I-CDs. It was found that the fluorescence intensity of N, S, I-CDs was proportional to the temperature in the range of 10e80 C (Fig. 7A). The fluorescence intensity had absolute dependence on the temperature in the process of varying the temperature. Fig. 7B showed the linear regression equation between I/I0 and temperature: I/I0 ¼ -0.01 t þ 1.0975 (R2 ¼ 0.9947). Obviously, I/I0 decreased by 68% when the temperature was raised from 10 C to 80 C. Also, it exhibited the accuracy of temperature measurement by N, S, I-CDs was high. The stability of temperature response towards N, S, I-CDs was tested by cyclic experiments. Temperature was changed continuously in the range of 10e80 C. After five cycles, as shown by Fig. 7C, it still maintained a good dependence. It showed that N, S, I-CDs gained good stability for temperature detection. The mechanism behind the response of N, S, I-CDs to temperature may be due to the increase in number of nonradiative channels on surface defects with raise in temperature. The increase in the number of non-radiative channels directly results in more electrons returning from the excited state to the ground state, thus weakening the fluorescence intensity. On the contrary, as the temperature decreases, the number of non-radiative channels decreases, and the electrons return to the excited state to restore the fluorescence intensity. Therefore, N, S, I-CDs are stable temperature detectors. Based on the above experimental results, the N, S, I-CDs can be a good fluorescent probe for biological imaging of cells too.
Fig. 6. Confocal microscopy images of U-2 OS incubated with 200 mg mL1 N,S,I-CDs (A1-A4) and after addition of 80 mM (B1-B4) and 175 mM (C1-C4) of FA in the culture solution (measured temperature: 25 C).
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Fig. 7. (A) Fluorescence intensity variation of N,S,I-CDs at different temperatures. (B) I/I0-temperature plots of N,S,I-CDs during 10e80 C. (C) The cycle experiment of the temperature response with N,S,I-CDs.
Therefore, based on the characteristics of N, S, I-CDs as a temperature sensor, we studied the temperature dependence of N, S, I-CDs in colon cancer cell HT-29. The confocal laser scanning microscopy image in Fig. 8 displayed the cell imaging of N, S, I-CDs and it exhibited a good cell morphology. We can see that the N, S, I-CDslabeled colon cancer cell HT-29 displayed an image with bright blue color spots at 15 C (Fig. 8A). As the temperature increases, the blue color spots were decreased in number at 25 C (Fig. 8B), and on further increase in temperature the blue color becomes significantly darker at 35 C (Fig. 8C). When the temperature was lowered to 15 C (Fig. 8D), the N, S, I-CDs-labeled colon cancer cell HT-29 returned to a bright blue color. More importantly, the fluorescence intensity of the labeled cells did not significantly decreased after 20 min of continuous excitation. The observed result confirms that N, S, I-CDs can be used in biomedical applications while performing the cell labelling and can be used in industries for temperature detection.
4. Conclusions In summary, N, S, I-CDs with high fluorescence quantum yield (32.4%) were successfully synthesized by one-step hydrothermal method using C3N3S3 and EDA as precursors, potassium iodate as additives to improve the fluorescence yield. A series of experiments were proved that N, S, I-CDs have good stability, hydrophilicity, low toxicity and good biocompatibility. This N, S, I-CDs displayed a significant response to the folic acid detection in the linear range of 0.1e175 mM with a promising detection limit of 84 nM. It had shown the stable temperature dependence emission in the range of 10e80 C, at the same time, stable reversibility of emission also seen. In addition, due to the less toxicity of N, S, I-CDs, they were successfully utilized as a nice cellular imaging agent in U-2 OS and colon cancer cell HT-29. Therefore, the synthesized N, S, I-CDs have a broad development in the field of biomedicine and environmental application after further development.
Fig. 8. Confocal microscopy images of N,S,I-CDs-colon cancer cell HT-29 with corresponding fluorescence field at 15, 25, 35 and 15 C, respectively.
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Acknowledgment This study was supported by the Analysis and Testing Foundation of Kunming University of Science and Technology (2018M20172118065), Yunnan Province, China. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.saa.2019.117444.
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