Research Article pubs.acs.org/journal/ascecg
Green Preparation of S and N Co-Doped Carbon Dots from Water Chestnut and Onion as Well as Their Use as an Off−On Fluorescent Probe for the Quantification and Imaging of Coenzyme A Yuefang Hu,†,‡ Liangliang Zhang,*,† Xuefeng Li,† Rongjun Liu,† Liyun Lin,† and Shulin Zhao*,† †
State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, College of Chemistry and pharmacy, Guangxi Normal University, Guilin 541004, China ‡ College of Materials and Environmental engineering, Hezhou University, Hezhou 542899, China S Supporting Information *
ABSTRACT: Fluorescent carbon dots (CDs) originated from natural biomass have been of great interest in recent years because of their superior optical and chemical properties. However, previously reported CDs used only one natural biomass as precursor, and the fluorescence quantum yield (QY) and long-wavelength emissions are usually weak, which restrict their further applications in biology-relevant fields. Here a green method was demonstrated for the preparation of S and N codoped fluorescent CDs (S,N/CDs) by adopting two natural biomasses (water chestnut and onion) as precursors. The fabrication process is simple and environmentally friendly. By hydrothermal heating of water chestnut and onion, monodispersed, highly fluorescent S,N/CDs (diameter 3.5 nm) were obtained. The carboxyl on the surface of S,N/CDs can bind to Cu(II) ion, resulting in the luminescence quenching of S,N/CDs. And coenzyme A (CoA) can restore the luminescence of S,N/CDs. Based on the above features of S,N/CDs, an innovative off−on fluorescence probe was presented for high sensitivity determination of CoA. Under optimum conditions, the linear range for CoA detection is 0.03−40 μM with a detection limit of 0.01 μM. The developed off−on nanoprobe was applied for the quantification of CoA in pig liver, and imaging of CoA in living T24 cells. KEYWORDS: S and N codoped carbon dots, Water chestnut, Onion, Coenzyme A detection, Fluorescent probe
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INTRODUCTION Luminescent carbon dots (CDs) as a new class of fluorescence material has been of great interest in the past ten years, owing to superior optical and chemical features, including adjustable size, strong resistance to photobleaching, high fluorescence stability, and good biocompatibility.1−5 The raw materials for CDs synthesis include mainly both inorganic and organic compounds, such as graphite, activated carbon, candle burning ash, carbon nanotubes, citric acid, ammonium citrate, and other carbon compounds.6 The preparation methods include generally carbonization of organic compounds, thermal decomposition, and microwave assisted preparation. However, using the above-mentioned methods for fabrication of CDs, still there are some disadvantages, such as low fluorescence quantum yield. Recently, heteroatom doping was supposed to be a promising method for improving the optical properties of CDs and increasing the quantum yield (QY) of CDs.7 Particularly, the nitrogen (N) atom can combine strongly with the C atom due to the size of it being similar to C; therefore, N-doped CDs (N/CDs) were shown to have higher fluorescence QYs,8,9 while sulfur (S) atom can adjust the density of states and afford emissive trap states (ETSs), which result in the electrons being excited to revise the band gap © 2017 American Chemical Society
energy. Therefore, S doping can tune the maximum fluorescence emission of CDs to a longer wavelength, and improve the fluorescence intensity of CDs.10 Then, S and N have been used as heteroatoms to synthesize S and N codoped CDs (S,N/CDs).11−13 However, many conventional methods for the preparation of these heteroatoms codoped CDs are expensive and ungreen. Recently, using natural biomass as a precursor to prepare fluorescent CDs has become interesting.14−17 Liu et al. prepared N-doped CDs by hydrothermal heating of pomelo peel.18 Zhao et al. used garlic as a precursor to prepare N and S codoped CDs.19 Li et al. prepared N-doped CDs from ginger.20 Sahu et al. used orange juice as a precursor to prepare N-doped CDs.21 And highly photoluminescent N-doped CDs derived from egg white have also been demonstrated by Zhang et al.22 This research has shown that natural biomass as a raw material has a great effect on the luminescence properties and biological activity of CDs. Thus, the S,N/CDs from various natural Received: February 8, 2017 Revised: April 6, 2017 Published: April 25, 2017 4992
DOI: 10.1021/acssuschemeng.7b00393 ACS Sustainable Chem. Eng. 2017, 5, 4992−5000
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centrifuged for another 10 min at 12,000 rpm under the same temperature. The obtained solution was then added into a cartridge (Sep-Pak C18), and the cartridge was washed sequentially with 40 mL of HCl solution (0.001 M), petroleum ether, and methanol. Finally, the CoA in the cartridge was eluted with a 6 mL mixture solution of C2H5OH and water (65:35 v/v) containing 0.10 M NH4Ac. The eluted CoA solution was evaporated by rotary evaporator to remove the ethanol, and diluted to an appropriate concentration with ultrapure water. Detection of CoA. The procedure for the fluorescence detection was as follows: 370 nm was adopted as the excitation wavelength. The S,N/CDs-Cu(II) probe was prepared by diluting 30 μL of S,N/CDs solution (10 μg/mL) and 20 μL of Cu(II) ion (30 μM) solution with 80 μL of 100 mM PBS solution (pH 5.8). After 6 min, 20 μL of CoA solution or pig liver sample solution was added into the above S,N/ CDs-Cu(II) probe solution, and the fluorescence spectrum of the system solution was measured after 1 min at room temperature. Cytotoxicity Experiment. The cells toxicity of S,N/CDs-Cu(II) probes was evaluate by MTT assay. Human bladder cancer T24 cells were incubated on a 96-well plate for 24 h with the density of 1 × 104 cells per each well. After removing extracellular medium, the S,N/ CDs-Cu(II) probe solutions with different concentrations (0, 10, 30, 50, 100, 300 μg/mL) were added into every well, and the mixture was incubated for 24 h. Then the cells were washed, and incubated another 4 h with fresh culture media (200 μL) containing 20% 3-(4,5)dimethylthiahiazo(-z-y1)-3,5-diphenyltetrazoliumro-mide, and the mixture was incubated a further 4 h. After removing the medium, 100 μL of dimethyl sulfoxide was added into every well, and the cells in the well were reacted with the dimethyl sulfoxide for 15 min. The optical measures of these wells were performed at 570 nm by an Enzyme Linked Immunosorbent Assay reader. Cells Culture and Imaging. The human bladder carcinoma cell line (T24 cells) was grown on a 35 mm glass culture dish for 24 h at 37 °C under a 5% CO2 atmosphere. For CoA imaging, 10 μg/mL S,N/ CDs solutions were added above the glass culture dish containing cells, and reacted with cells for 6 h at 37 °C. After removing extracellular medium, the cells were washed three times with PBS, and incubated with Cu(II) (30 μM) at 37 °C for 30 min. In the control group, after removing extracellular medium, 15 mM N-ethylmaleimide was added into the glass culture dish containing cells, and the cells were cultured for 30 min to consume all free thiols in the cells, and then incubated with S,N/CDs-Cu(II) solution for 30 min. To verify the identification of CoA in cells, the sample was spiked with 150 μM CoA and imaged again. First, the cells were washed twice with Dulbecco’s modified eagle medium (DMEM) solution, and pretreated with 150 μM CoA for 30 min, and the cells were washed again three times with PBS solution. Finally the cells were incubated with S,N/CDs-Cu(II) solution for a further 30 min at 37 °C, and measured by confocal imaging after washing the cells three times with PBS solution.
biomasses are desired for obtaining CDs with higher fluorescence QY and biological activity. The coenzyme A (CoA) is a kind of important coenzyme,23 and plays important roles in many biochemical reactions of the human body, such as neurodegeneration, protein acetylation, autophagy, and signal transduction.24 The deficiency of CoA could disturb the biological processes of the human body and cause different damages.25,26 Thus, CoA is often used as a clinical medicine to improve the following symptoms, including irritability, anxiety, fatigue, and anesthesia. Therefore, the detection of CoA in biological samples is essential for understanding the pathogenesis of diseases, and developing sensitive, accurate, and simple detection methods for CoA is highly desirable in biomedical and bioscience studies. The water chestnut contains many nitrogenous compounds, and onion contains a large number of thiol compounds. Herein, we use water chestnut as N source and onion as S source for green fabrication of S,N/CDs, and we found that the carboxyl on the S,N/CDs surface can bind to Cu(II) ion, resulting in the luminescence quenching of S,N/CDs. And the CoA can restore the fluorescence of S,N/CDs. Then, we further use S,N/CDs as a sensitive fluorescence probe for the quantification and imaging of CoA.
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EXPERIMENTAL SECTION
Materials and Chemicals. The water chestnut and onion were bought from a vegetable market (Hezhou, China). CoA, Nethylmaleimide, and other chemicals used in this work were bought from Aladdin Chemistry Co. Ltd. (Shanghai, China). The solutions used in this work were prepared in ultrapure water, which was prepared using a water purification system (Millipore, Bedford, MA). Apparatus. The apparatuses for the structure and composition characterization of S,N/CDs are the same as that used in previous work.27,28 Preparation of S,N/CDs. The S,N/CDs were synthesized by heating the water chestnut and onion in an autoclave. Briefly, 2 g of fresh water chestnut and 3 g of fresh onion were crushed into a powder, and transferred to a 50 mL Teflon-lined autoclave. Then 30 mL of ultrapure water was added into the Teflon-lined autoclave, and the autoclave was heated in a muffle furnace for 4 h at 180 °C. The resultant brown mixture was collected by centrifugation for 20 min at 12,000 rpm. The collected brown mixture was dialyzed for 48 h through a dialysis membrane (MWCO = 1000) in ultrapure water. The dialysate was stored at 4 °C for S,N/CDs solution. Determination of QY. The QY of S,N/CDs was detected by using quinine sulfate as reference substance, and obtained by the following equation:
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Φ = Φref × (Isam/Iref ) × (A ref /A sam ) × (η2 sam /η2 ref )
RESULTS AND DISCUSSION Synthesis of S,N/CDs. In order to get a suitable emission wavelength and QY, the reaction conditions, including the mass ratios of water chestnut to onion, the reaction time, and temperature, were optimized. The experimental results indicate that the QY rises with the increase of the mass ratios of water chestnut to onion from 1:4 to 3:2; however, the maximum emission wavelength has a blue-shift when the mass of water chestnut (Table S1) increases. Considering the long emission wavelength and high QY for S,N/CDs, the optimal mass ratios of water chestnut to onion was set as 2:3 for the subsequent experiment. The reaction temperature and time also affect the average size and QY of S,N/CDs. The higher reaction temperature and shorter reaction time are favorable to obtain the small size of S,N/CDs (Table S2). Therefore, the 180 °C and 4 h were selected for optimal reaction temperature and time.
In the above equation, Φ and I are the QY and integrated emission intensity. A and η are the optical density and refractive index. The subscripts “ref” and “sam” are the reference substance with known Φ and the samples, respectively. Fluorescence Lifetime. The time-resolved fluorescence decay curve was used to evaluate the fluorescence lifetime, and the data were obtained by the following equation: I(t) = A + B1 exp(−t/τ1) + B2 exp(−t/τ2). In the formula, B represents the relative amplitude, and τ represents the component lifetime. Average fluorescence lifetime ⟨τ⟩ was obtained from the below formula:
⟨τ ⟩ = B1τ1/(B1 + B2 ) + B2 τ2/(B1 + B2 ) Pretreatment of Liver Samples. Fresh pig livers were obtained from a local supermarket. First, 4 g of pig liver sample was cut into liver homogenates, and mixed with 20 mL of 8.4% HClO4. The mixture solution was centrifuged at 18,000 rpm for 5 min. The supernatant solution was adjusted to pH 3.0 by adding 20% KOH, and 4993
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ACS Sustainable Chemistry & Engineering Structure and Composition of S,N/CDs. The TEM image of S,N/CDs presents a good dispersibility and uniformity (Figure 1a). The HRTEM image displays two lattice spacings,
using water chestnut as precursor without onion under the same conditions. The N/CDs solution displays blue luminescence under irradiation at 370 nm; the maximum emission wavelength (440 nm) of N/CDs was shorter than that (475 nm) of S,N/CDs (Figure S2). It was confirmed that the green-blue fluorescence emission of prepared S,N/CDs derives indeed from doped S atom. The fluorescence spectra of the S,N/CDs under the excitation of different wavelengths of light (330−470 nm) were shown in Figure 4b. As can be seen, the fluorescence emission of the S,N/CDs is dependent on the excitation light. This excitation-dependent phenomenon is due to the difference in particle size and the distribution of diverse emissive trap sites of the S,N/CDs.17 The optical properties under various conditions were also investigated. The experimental results show that the S,N/CDs also exhibit good photostability and are dispersible in water. When the S,N/CDs were irradiated continuously under 365 nm excitation for 3 h, or preserved in solution for 6 months at room temperature, the fluorescence intensity was not changed (Figure S3a), which indicates that the S,N/CDs have the excellent property of resistance to photobleaching, and the fluorescent spectra of S,N/CDs at various pH values show a different change as the pH increases from 2 to 13 (Figure S3b). Using quinine sulfate as the reference, the QY of prepared S,N/CDs was obtained to be 12%, which is much higher than that of many reported CDs that were prepared using single biomass as precursor, including coffe grounds (3.8%),15 watermelon peel (6.7%),16 and sweet potatoes (2.8%).33 The reasons for the higher QY using two kinds of biomass as precursors simultaneously may be owing to the synergistic effects between N atom in water chestnut and S atom in onion. And two kinds of biomass heating together may cause a complex reaction and produce complex polymer mixtures. The polymer mixtures are rich in different functional groups, including −OH and −NH2, which can generate many defects on the surface of S,N/CDs, serve as the excitation energy traps, and result in excellent PL properties.34 Mechanism of Off−On Fluorescence for CoA Detection. Scheme 1 shows the mechanism of fluorescence “off” and “on” using S,N/CDs-Cu(II) as fluorescence probes for CoA detection. As can be seen, S,N/CDs have a strong green-blue fluorescence emission under 370 nm light excitation. After adding Cu(II) ion, the green-blue fluorescence was quenched dramatically. This result verifies that the luminescence of S,N/ CDs can be quenched by Cu(II) ion because Cu(II) ion chelated easily with the carboxyl and hydroxyl groups of the S,N/CDs surface, and formed a nonradiative complex (S,N/ CDs-Cu(II)). The form of the nonradiative complex changed the defects and the distribution of excitons on the surface of S,N/CDs, which resulted in the fluorescence quenching to reach the “off” process. After adding CoA, Cu(II) ion was detached from the S,N/CDs-Cu(II) complex due to the thiol group in CoA molecule strongly binding toward Cu(II) ion to form a more stable Cu(II)−S bond and result in the recovery of S,N/CDs luminescence to display the “on” state. To confirm the above mechanism, the fluorescence spectra of the S,N/CDs, S,N/CDs-Cu(II), and S,N/CDs-Cu(II) + CoA systems under 370 nm light excitation were examined. It was found that the maximum emission of the S,N/CDs-Cu(II) + CoA system wavelength does not shift with the fluorescence recovery of the system (Figure 5a), which further indicated that
Figure 1. TEM and HRTEM (inset) images of S,N/CDs (a), and the size distribution of S,N/CDs (b).
0.23 and 0.32 nm, which is consistent with the (100) and (002) diffraction facets of graphite diffraction (inset in Figure 1a).11 Figure 1b was received by counting about 100 particles, and counting results manifest the prepared S,N/CDs have smaller size, with diameters in the range 2.0−4.0 nm and average size 3.5 nm. The Raman spectrum of S,N/CDs (Figure S1) displays two bands at 1354 and 1574 cm−1, which represent the D and G bands of S,N/CDs, respectively.29 This information indicates that S,N/CDs were composed of a graphitic sp2 carbon atom and sp3 carbon defects. The surface-state of the S,N/CDs was validated by XPS (Figure 2). The obtained information concerning typical peaks is detailed in the Supporting Information. The surface groups of the S,N/CDs were confirmed by FTIR. Obtained FT-IR spectral (Figure 3) information is also detailed in the Supporting Information. Photoluminescence Features of S,N/CDs. To investigate the photoluminescence (PL) features of S,N/CDs, PL and UV−vis spectra of S,N/CDs were measured. As can be seen in Figure 4a, two representative peaks at 242 and 333 nm are shown in the UV−vis spectrum of S,N/CDs. The peak at 242 nm is assigned to the π → π* transition of aromatic sp2 domains.30 The peak at 333 nm is from the trapping of excited state energy.31 As expected, strong green-blue emission in the range 400−600 nm appeared in S,N/CDs solution under 370 nm light excitation (inset in Figure 4a) and shows the maximum emission at 475 nm (Figure 4a). This green-blue emission of S,N/CDs is due to doping of S atom.15,32 To further verify this explanation, the N/CDs were prepared by 4994
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Figure 2. XPS spectra of the S,N/CDs (a) and high resolution C 1s peak, N 1s peak, and S 2p peak of S,N/CDs (b, c, d).
Figure 3. FT-IR spectra of S,N/CDs.
the restoration of the quenched fluorescence is due to the interaction between Cu(II) ion and CoA. The fluorescence lifetimes of S,N/CDs in the presence of different concentrations of Cu(II) ion were examined to further confirm the above quenching mechanism. If the fluorescence lifetime was decreased after adding the quencher, the system is dynamic quenching. If the fluorescence lifetime was unchanged (τ0/τ = 1), the system is static quenching.35 Figure 5b shows the fluorescence decay curves of S,N/CDs in the presence of different concentrations of Cu(II) ion. The fluorescence lifetime of S,N/CDs was calculated to be 5.08 ns (τ1 = 2.67, τ2 = 7.48), which is longer than that of the CQDs (2.8 ns). It was reported that the doping of N and S can bring in localized states in the CQDs, which could trap photoexcited electrons and prolong the fluorescence lifetime.36 After gradually adding Cu(II) ion from 0 to 30 μM in the above system, the average lifetime of S,N/CDs only decays to 4.78 ns (τ1 = 2.38, τ2 =
Figure 4. UV−vis absorption spectra and fluorescence spectra of S,N/ CDs, inset: photograph of the S,N/CDs solution under 370 nm excitation (a). Fluorescence emission spectra for S,N/CDs solution under different excitation wavelengths (b).
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considering the low background signals and wide linear range, 30 μM was selected for optimal Cu(II) ion concentration.
Scheme 1. Schematic Illustrations for the Synthesis of the S,N/CDs-Cu(II) Probe and the Detection of CoA
Figure 6. Fluorescence spectra of S,N/CDs in the presence of different concentrations of Cu(II) ion. Inset: The plot I/I0 versus the concentration of Cu(II) ion. I0 and I are the fluorescence intensity of S,N/CDs in the absence and presence of Cu(II) ion, respectively.
The pH value of the reaction solution was optimized, and the ratios (I0/I) of the fluorescence intensities of S,N/CDs and S,N/CDs-Cu(II) under different pH values ranging from 4.5 to 7.5 were examined. It was found that the largest ratio was obtained at pH 5.8 (Figure S4a). Meanwhile, the ratios (I′/I′0) of the fluorescence intensities of S,N/CDs-Cu(II) solution under different pH values ranging from 4.5 to 7.5 were also examined in the presence and absence of CoA. The maximum value of I′/I′0 was also found at pH 5.8 (Figure S4a), in which I′ and I′0 represented the fluorescence signals of S,N/CDsCu(II) with and without CoA. Therefore, the pH 5.8 was selected as the optimal reaction acidity for the off−on system. The influence of irradiation time on the stability of the fluorescence signal of S,N/CDs, S,N/CDs-Cu(II) and S,N/ CDs-Cu(II) + CoA systems were also investigated. The fluorescence signal was recorded with the irradiation time from 1 to 40 min (Figure S4b). The test results shows that the fluorescence signal of S,N/CDs keeps unchange within the range of 1−40 min. After adding Cu(II) ion, the fluorescence signal decreased gradually and reached equilibrium at about 6 min, and remained constant after 6 min. Satisfyingly, the quenched fluorescence for the S,N/CDs-Cu(II) system was recovered rapidly by adding CoA, the fluorescence signal reached the highest after 1 min, and almost remain invariableness within 40 min at room temperature. Linear Range and Limit of Detection. The proposed method for CoA detection was evaluated by the response linearity to CoA and the detection limit. As can be seen in Figure 7a, the fluorescence signal of S,N/CDs was restored increasingly with increasing concentration of CoA. The enhancement value of the fluorescence intensity of the system shows a linear relationship with increasing CoA concentration from 0.03 μM to 40 μM (Figure 7b). The linear regression equation was I′/I′0 = 0.1333C (μM) + 1.196, r = 0.9970. Based on signal/noise = 3, the detection limit was calculated to be 0.01 μM. Compared with reported methods in the literatures for the determination of CoA (Table S3), the proposed fluorescence probe has several features, such as high detection sensitivity, wide detection range, and no chemical modification, which shows great promise in biomedical and clinical diagnosis assays.
Figure 5. Fluorescence spectra of S,N/CDs, S,N/CDs-Cu(II), and S,N/CDs-Cu(II) + CoA (a), and time-resolved fluorescence decay spectrum of S,N/CDs by adding different amounts of Cu(II) ion (b).
7.39). As can be seen from Figure 5b, the decay curves of them come near to each other. Minor fluorescence lifetime change has proved that the Cu(II) quenching the fluorescence of S,N/ CDs is a static quenching process because of forming a stable nonradiative complex.37 Optimization of Reaction Conditions for CoA Detection. To achieve a highly sensitive CoA assay, we optimized the reaction conditions for CoA detection including the concentrations of Cu(II) ion, the acidity of the reaction solution, as well as the incubation time. The quenching effect of Cu(II) ion with different concentrations was investigated. The result indicated that the fluorescence signal of the S,N/CDs was decreased increasingly when the concentrations of Cu(II) ion increased from 0 to 30 μM, and after the concentration was more than 30 μM, the fluorescence intensity only decreased slightly (Figure 6). Thus, 4996
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Table 1. Content, Recoveries, and RSDs of CoA in Pig Liver Samples (n = 6) Sample Sample 1
Sample 2
Sample 3
Added (10−7 mol g−1)
Found (10−7 mol g−1)
Recovery (%)
RSD (%)
0 1.00 1.50 0 1.00 1.50 0 1.00 1.50
1.38 2.51 3.02 1.35 2.49 3.00 1.26 2.39 2.95
101.0 99.7 99.5 102.2 101.0 102.5
1.4 2.5 2.3 1.6 2.7 2.4 2.1 2.5 3.0
bladder cancer T24 cells with an MTT test.40 As shown in Figure 8, the S,N/CDs-Cu(II) probe did not show apparent
Figure 7. Fluorescence response of S,N/CDs-Cu(II) probe upon addition of various concentrations of CoA. CoA concentrations (from bottom to top) are 0, 0.03, 0.8, 8, 15, 23, 26, 31, 40, 60, 100, and 150 μM (a). Plot of I′/I′0 of S,N/CDs-Cu(II) vs the concentration of CoA (b). Figure 8. Cell viability after incubation with S,N/CDs-Cu(II) for 24 h.
Selectivity Study. In order to assess the selectivity of the S,N/CDs-Cu(II) probe toward CoA, the fluorescence responses of S,N/CDs-Cu(II) to possible coexistence substances, including biothiols (GSH, Hcy, and Cys), amino acids, anions, glucose, sucrose, lactose, uric acid, guanine, adenosine, adenosine triphosphate (ATP), and dopamine (DA), were investigated. Figure S5 shows that the anions, glucose, sucrose, lactose, uric acid, guanine, adenosine, adenosine ATP, DA, and most amino acids do not affect the detection of CoA. Some biothiols can only partially recover the fluorescence, but the I′/ I′0 value was far below that of CoA. The CoA as an acid has a lower pKa value (6.40) compared to that of GSH (9.20), Hcy (8.87), and Cys (8.00). Therefore, it can be used for specific detection of CoA without any interference from the above three biothiols at pH 5.8. The thiolate is the primary form of CoA at pH 5.8, and serves as a more preferential coordinating ligand with Cu(II) ion than GSH, Cys, and Hcy.38 Therefore, the influence from biothiols was negligible in the CoA detection, which demonstrates that the proposed S,N/CDsCu(II) probe has excellent selectivity and can meet the selective requirement for CoA detection and imaging. Detection of CoA in Pig Liver. Three pig liver samples were analyzed to assess the applicability of the constructed fluorescent probe. As shown in Table 1, the CoA level in three pig liver samples was found be in the range 0.12−0.14 μM. The content of CoA in pig liver is similar to that of previous reports.39 Recoveries of CoA were found in the range 99.5− 102.5%, and the relative standard deviations (RSDs) were lower than 3%. These results indicate that the constructed method was feasible for determination of CoA in pig liver samples. Cytotoxicity Testing. The cell cytotoxicity experiment of the S,N/CDs-Cu(II) probe was evaluated by using human
cytotoxicity even though the S,N/CDs-Cu(II) probe concentration was increased to 300 μg/mL. This result suggests that the S,N/CDs-Cu(II) probe has low cytotoxicity and excellent biocompatibility, and can be effectively applied for bioimaging and cell tracking. Imaging of CoA in Living Cells. The imaging of CoA in the T24 cells was studied by using the S,N/CDs-Cu(II) as a fluorescence probe. Figure 9 shows the laser confocal fluorescence images of T24 cells excited at 405 and 488 nm. After the S,N/CDs-Cu(II) probe was loaded into the T24 cells medium for 30 min, blue and green fluorescence was found in the cells under 405 and 488 nm light exciting (Figure 9A), which demonstrate that the S,N/CDs-Cu(II) probe can penetrate cell membranes and image intracellular CoA. However, the T24 cells were pretreated with 15 mM Nethylmaleimide, which can consume all thiols within the cell through Michael addition,41 and then incubated with a S,N/ CDs-Cu(II) probe under the same conditions. No obvious fluorescence was observed in the T24 cells (Figure 9B). While the T24 cells were pretreated with 150 μM CoA for 30 min, and then incubated with a S,N/CDs-Cu(II) probe for another 30 min, stronger blue and green fluorescence was observed in the T24 cells (Figure 9C) compared with that shown in Figure 9A, which indicates that the proposed S,N/CDs-Cu(II) probe has excellent selectivity and is suitable for imaging of CoA in living cells.
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CONCLUSIONS In this work, we have developed a green strategy for the fabrication of S,N/CDs by using two natural biomasses (water 4997
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Figure 9. Laser confocal fluorescence images of T24 cells excitation at 405 and 488 nm. (A) Incubated with S,N/CDs-Cu(II). (B) Pretreated with N-ethylmaleimide, and then incubated with S,N/CDs-Cu(II). (C) Pretreated with CoA and then incubated with S,N/CDs-Cu(II).
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chestnut and onion) as precursors. The fabrication process is simple, low-cost, and environmentally friendly. The obtained S,N/CDs are nanometer-sized, about 3.5 nm, and exhibit good dispersibility and strong fluorescence. The fluorescence of S,N/ CDs could be quenched by Cu(II) ion and restored by CoA. These S,N/CDs have almost no cytotoxicity. Based on the optical and chemical properties of S,N/CDs, a new off−on fluorescence probe was presented for high sensitivity quantification of CoA in pig liver, and imaging of CoA in living cells. We expect that this novel nanoprobe would be a powerful tool for imaging endogenous CoA and finding new physiological functions of CoA in living cells.
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AUTHOR INFORMATION
Corresponding Authors
*Shulin Zhao. E-mail:
[email protected]. *Liangliang Zhang. E-mail:
[email protected]. ORCID
Shulin Zhao: 0000-0002-2560-042X Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundations of China (21327007, 21575031), the Natural Science Found ations of Guangxi Province (No. 2015GXNSFDA 139006, 2014GXNSFBA118047), and the BAGUI Scholar Program.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b00393. Raman spectroscopy of the S,N/CDs, fluorescence spectra of S,N/CDs and N/CDs excited at 370 nm light, effect of irradiation time at 365 nm light and solution pH value on the fluorescence signal of S,N/CDs, effect of the solution pH value on I0/I and I′/I′0, effect of incubation time on the fluorescence response of the S,N/ CDs, S,N/CDs-Cu(II), and S,N/CDs-Cu(II)+CoA systems, fluorescence response of the S,N/CDs-Cu(II) probe with 150 μM possible coexistence substances, fluorescence spectra of S,N/CDs synthesized by different mass ratios of water chestnut and onion, effects of different reaction times and temperatures on the size and quantum yield of S,N/CDs, and comparison of some methods used for CoA detection (PDF)
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DOI: 10.1021/acssuschemeng.7b00393 ACS Sustainable Chem. Eng. 2017, 5, 4992−5000
