Research Article www.acsami.org
Red Emissive Sulfur, Nitrogen Codoped Carbon Dots and Their Application in Ion Detection and Theraonostics Xiang Miao,†,‡,⊥ Xinlong Yan,*,§ Dan Qu,†,‡,⊥ Dabing Li,† Franklin Feng Tao,∥ and Zaicheng Sun*,‡ †
State Key Laboratory of Luminescence and Applications, Changchun Institute of Optics, Fine Mechanics and Physics, Changchun, 130033 Jilin, People’s Republic of China ‡ Beijing Key Laboratory for Green Catalysis and Separation, Department of Chemistry and Chemical Engineering, School of Environmental and Energy, Beijing University of Technology 100 Pingleyuan, Chaoyang District, Beijing 100124, People’s Republic of China § College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, People’s Republic of China ∥ Department of Chemical and Petroleum Engineering, Department of Chemistry, University of Kansas, Lawrence, Kansas 66047, United States ⊥ University of Chinese Academy of Sciences, Beijing 100000, People’s Republic of China S Supporting Information *
ABSTRACT: It is highly desirable and a great challenge for red light emission of carbon dots under long wavelength excitation. Here, we developed a facile route to synthesize carbon dots with red emission due to the doping effect of S and N elements, borrowing from the concept of the semiconductor. The maximum emission locates at 594 nm under 560 nm excitation. The absolute photoluminescence (PL) quantum yield (QY) is as high as 29% and 22% in ethanol and water, respectively. XPS and FTIR spectra illustrated that there exist -SCN and -COOH groups on the surface of the carbon dots. They endow the carbon dots with high sensitivity for ion detection of Fe3+. The quenched PL emission of Fe3+-S,N-CDs can be recovered by adding ascorbic acid to release the -COOH and -SCN group due to Fe2+ formation in the presence of ascorbic acid. High PL QY of red emission is beneficial to application in bioimaging. Doxorubicin was loaded onto carbon dots through π−π stacking to form a theranostic agent. When the CD-Dox was injected into the tumor site, a strong PL emission was observed. The PL intensity indicates the concentration of the theranostic agent. After 7 times injection, both the tumor size and weight clearly decrease. The results demonstrate that the S,N-CDs are a potentially excellent bioimaging component in the theranostic field. KEYWORDS: S,N codoped carbon dots, red light emission, fluorescent sensor, theranostic agent, nanomedicine
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INTRODUCTION Carbon dots (CDs) have been treated as a promising fluorescent material due to their attractive properties including excitation-dependent multiple color emission, less toxicity, excellent biocompatibility, and multiple functional groups, etc.1−3 CDs have been developed for more and more applications in the optoelectronic conversion,4−7 bioimaging,8,9 detection,10−13 nanomedicine,14−16 and catalyst.17−20 Among reported CDs, excitation-dependent multiple color emission is the most attractive property. However, most of the known CDs have maximum blue light emission, which limits its biorelated © 2017 American Chemical Society
application due to the short wavelength excitation light being harmful to the living cells and biological systems.21 Such drawbacks severely hampered their further application in the fields of biology, because of the well-known blue autofluorescence of a biological system and photodamage of UV excitation light. Therefore, it is highly desirable and a great challenge to achieve highly efficient red light emissive CDs, Received: March 30, 2017 Accepted: May 16, 2017 Published: May 16, 2017 18549
DOI: 10.1021/acsami.7b04514 ACS Appl. Mater. Interfaces 2017, 9, 18549−18556
ACS Applied Materials & Interfaces
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Research Article
RESULTS AND DISCUSSION Characterizations of S,N-CDs. Typically, highly fluorescent S,N-CDs were prepared with citric acid (CA; 3 mmol) and thiourea (TU; 10 mmol) in 10 mL of acetone via hydrothermal route. The S,N-CDs dispersion in water is a transparent and homogeneous solution even after storage for several months at room temperature. As shown in Figure 1a, the transmission
which can be excited under long wavelength light irradiation and has better organ penetration depth. To our best knowledge, only a few reports have demonstrated red emissive CDs with relatively low photoluminescent (PL) quantum yields (QYs) and a complicated synthetic route. Recently, Ge et al. showed red emissive CDs prepared from polythiophene derivatives as a carbon source. However, multiple reaction steps were needed to obtain the starting materials and the PL QY of as-prepared CDs is very low (2.3%).22 Our group demonstrated CD with three colors emission using citric acid and thiourea as sources through a hydrothermal route in N,N-dimethylformide solution.23 However, the PL QY of CDs is quite low (8%) in the red light emission. Furthermore, Lin’s group used phenyldiamine as carbon and nitrogen source to obtain N doped CDs with three color light emissions, of which PL QY of red emissive CDs reaches 26%.24 Later, this group reported the CDs with red light emission prepared from citric acid in formamide solution under microwave assisted hydrothermal route. The PL QY of as-prepared CDs reaches 23%.25,26 Xiong’s group showed the red emissive CDs prepared from p-phenyldiamine and urea followed with silica column chromatography separation to get the right fraction. As they pointed out, it should be noted that the operation of the silica column chromatography apparatus required great patience and attentiveness.27 Very recently, Yang’s group reported red emission carbon dot prepared from dopamine and o-phenyldiamine by soaking reactor after hydrothermal reaction.28 There is no simple and facile route to obtain highly efficient red emissive CDs, although it is highly desirable for biorelated applications. For inorganic semiconductor, fluorescence is the emission of electron transition from the conduction band (CB) to valence band (VB). The bandgap of the semiconductor determines the emission wavelength. According to the band engineering theory, the doping process is an important method to tune the optical properties of semiconductors.29 In addition, a narrow band gap will result in long wavelength emission. Based on this, we proposed that doping heteroatom S may further narrow the band gap of N doped CDs because S has a lower electronegativity than O or N. Besides that, we found the reaction solvent also has a strong effect on the optical properties of CDs,23,30,31because solvent may take part in the reaction and introduce a special group on the CDs. In this report, a one-step hydrothermal route for preparation of S, N doped CDs (S,N-CDs) with red light emission is developed using citric acid and thiourea in acetone at 160 °C for 8 h. We found that S,N-CDs possess unique -SCN and -COOH groups, which could serve as an active site for the metal ion (Fe3+) detection. S,N-CDs show high sensitivity and selectivity on the Fe3+ with 9.7 nM (∼0.5 ppm) of limit of detection, which is more sensitive than the traditional organic molecules method of detecting iron ions.32 On the other hand, the maximum excitation and emission wavelength shift to ca. 560 and 610 nm, respectively, due to heteroatom doping effect. The red emission S,N-CD has a decent PL QY (29% in ethanol and 22% in water) and long lifetime (12.9 ns), which is a potential bioimaging agent for construction of theranostic nanomedicine. S,N-CDs/Dox theranostic agent was successfully fabricated by simply π−π stacking interaction between S,N-CDs and Dox. The results of in vitro and in vivo analysis demonstrated that the S,N-CDs/Dox has both imaging and therapeutic function.
Figure 1. TEM (a) and HR TEM (b) images of S,N-CDs. Insets of panels a and b are the particle size distribution of S,N-CDs and fast Fourier transform (FFT). Raman (c) and FT-IR (d) spectra of S,NCDs.
electron microscopy (TEM) images of S,N-CDs exhibit uniform nanoparticles with a diameter of 4.42 ± 0.67 nm. High-resolution TEM (HR-TEM) images disclose that most CDs have clear lattice structures with fringe spacing of 0.24 nm (Figure 1b), which is associated with the basal spacing of (1120) lattice plane of graphene.33 The corresponding fast Fourier transform (FFT) pattern exhibits hexagonal lattice indicating that the S,N-CDs are crystalline hexagonal structures. As shown in Figure S1 in the Supporting Information (SI), the X-ray diffraction (XRD) patterns of the S,N-CDs show one broad diffraction peak centered at 2θ = 22.6°, corresponding to d spacing of 0.34 nm. It can be indexed to the (002) lattice space of the graphite indicating that the S,N-CDs have a graphite-like structure. The graphitization is also confirmed by Raman spectrum (Figure 1c), where the signals at 1578 and 1346 cm−1 are assigned to the G band and disordered D band. The ratio of G to D bands is greater than 1, which manifests evidence the CDs have high graphitization. The surface functional groups on the surface of S,N-CDs were characterized through Fourier transform infrared (FTIR) spectroscopy (Figure 1d). The broad vibration bands at ∼3420 and ∼3200 cm−1 are attributed to stretching vibrations of O−H and N−H; the peaks at 1703 and 1655 cm−1 are ascribed to the νCO in the COOH and CONH2 group, respectively. The peaks at 1574 and 1384 cm−1 are attributed to the bending vibration of CC/N−H and O−H, respectively. The peaks at 1490 and 1453 cm−1 are assigned to δC−H and νC−N, respectively. The above results confirm the existence of -COOH, -OH, -NH2, and -CONH2 groups on the surface of 18550
DOI: 10.1021/acsami.7b04514 ACS Appl. Mater. Interfaces 2017, 9, 18549−18556
Research Article
ACS Applied Materials & Interfaces
energy (167.9 eV) is assigned to -C−SOx−C− sulfone bridges.36 Based on the XPS analysis, it is confirmed that the S,N-CDs are doped by N and S elements and functional groups such as -OH, -COOH, -NH2, and -SCN coexist on the CD’s surface. Through measuring the ζ potential (+21.7 mV) of the CDs in aqueous solution, it indicates that there are a lot of -OH and -NH2 on the surface of the CDs. The presence of hydrophilic groups is advantageous for the dispersion of S,NCDs in aqueous solution and may be employed as functional groups for detection of metal ions. Optical Properties of S,N-CDs. The UV−vis absorption spectrum (Figure 3a) shows absorption bands at 238, 279, and 360 nm results from π−π*transition of graphite sp2 domain and n−π* transition of CX (X = O, N) in the doped CDs, respectively.11,37 Besides these adsorption bands, two other adsorption bands at ∼464 and ∼562 nm are observed. A similar adsorption band is also observed in the S,N-CDs prepared from CA and TU in the DMF solution.23 The band at 464 nm is attributed to the n → π* transition of the conjugated CN bonds, and the band at 562 nm is mainly assigned to the n → π* transition of the conjugated CS bond. The PL spectra excited with different wavelengths are shown in Figure 3b. The S,N-CDs show weak wavelength-dependent emission and the maximum PL emission at ∼594 nm with excitation at ∼560 nm wavelength. The PL excitation spectrum is also displayed in Figure 3b; the excitation peak locates at 560 nm, which corresponds to the adsorption band at 562 nm. The longer excitation/emission wavelengths provide a great opportunity to have large tissue penetration depth for further biorelated applications. Furthermore, the absolute PL QYs of S,N-CDs in ethanol and water were as high as 29% and 22% for the emission at 594 nm, respectively. The fluorescence stability of CDs at different ionic strengths and pHs was investigated. Figure S2 shows CDs remained stable at different ionic strengths and the photoluminescent intensity or peak of the CDs did not change, which is very critical because there usually have high salt concentration in practical applications. PL intensities almost maintain constant in a low-pH solution (pH = 1−8) and decrease in a solution of high pH (pH = 8−14) (Figure S3). Ion Detection. Fe3+ ion is an irreplaceable element in the living system, and it is needed for certain physiological functions. It could be beneficial to detect Fe3+ through a visible fluorescent method. It has been demonstrated that the organic molecules fluorescence could be quenched by Fe3+, because Fe3+ can be coordinated with the functional groups such as phenolic hydroxyl and carboxylic groups, which has
CDs. Besides those peaks, a new strong peak appears at 2050 cm−1 in the FTIR spectrum, which can be assigned to the -SCN group. Furthermore, the element composition and valence state of S,N-CDs were characterized by X-ray photoelectron spectroscopy (XPS) technique. Figure 2a shows the full-scan survey of
Figure 2. Full-survey X-ray photoelectron spectroscopy (XPS) (a), C 1s (b), N 1s (c), and S 2p (d) XPS spectra of S,N-CDs.
S,N-CDs. The four main peaks at 532, 400, 284, and 162 eV represent O 1s, N 1s, C 1s, and S 2p XPS characteristic peaks, respectively. Figure 2b shows the high-resolution C 1s XPS spectrum, which consists of four peaks, corresponding to sp2 hybridized carbon atoms in C−C/CC (∼284.3 eV), sp3 hybridized carbon atoms in C−S/C−N/C−O (285.10 eV). -CN carbon at 286.1 eV, and a carboxylic group (COOH) at 288.5 eV. Comparing with our previous report,23 there existed no component at 288.5 eV associated with the carboxylic group in the C 1s XPS spectra when the S,N-CDs were prepared in DMF. The high-resolution N 1s XPS spectrum of S,N-CDs (Figure 2c) confirm the presence of pyrrolic N (399.5 eV) and graphite N (400.2 eV) atoms. Besides those, a peak at 397.1 eV, which is assigned to the -CN group, is observed. That indicates that -CN exists in the S,N-CDs. The S 2p XPS spectrum (Figure 2d) of CDs has three peaks: the peaks at 161.7 and 163.3 eV correspond to S 2p3/2and S 2p1/2spectra of the C−S−C covalent bond in thiophene-type structure owing to the spin−orbit splitting,34,35 while another peak at higher
Figure 3. UV−vis spectrum (a), photoluminescent (PL) spectra (b) under different excitation wavelength and excitation spectra of emission at 594 nm of S,N-CDs. The inset is the optical graph of S,N-CDs under day light (a) and excitation with green light (b). 18551
DOI: 10.1021/acsami.7b04514 ACS Appl. Mater. Interfaces 2017, 9, 18549−18556
Research Article
ACS Applied Materials & Interfaces
Figure 4. (a) PL emission quenching spectra of S,N-CDs with different concentrations of [Fe3+]. (b) Stern−Volmer plot of S,N-CDs quenched by Fe3+. (c) PL emission change of S,N-CDs in the presence of different metal ions. (d) Fluorescence spectra of Fe3+-S,N-CDs mixture with the addition of different amount of ascorbic acid concentration (0.2−2.0 μmol/L). (e) Lifetime decay of S,N-CDs and Fe3+-S,N-CDs. (f) Normalized fluorescent intensity of S,N-CDs, Fe3+-S,N-CDs, and Fe3+-S,N-CDs with excess amounts of AA and EDTA.
been applied to the detection of Fe3+ and organic colored reactions.11,38−40 Comparing with traditional Fe3+ sensor, the red emission S,N-CDs has longer excitation wavelength (∼560 nm), which avoids interference from the adsorption of Fe3+. Figure 4a is the fluorescent quenching spectra of S,N-CDs with different concentrations of Fe3+ ions. The PL intensity continuously decreases with the concentration of Fe3+. The intensity ratio of Fe3+-S,N-CDs vs the concentration of Fe3+ plot (Figure 4b) can be fitted into (I0/I) = 1.97 × 105[Fe3+] + 0.979, which is similar to the Stern−Volmer equation: (I0/I) = KSV[Fe3+] + 1, where I0 and I are the fluorescence intensities before and after the addition of Fe3+, respectively; and KSV is the Stern−Volmer constant. The KSV value is obtained to be 1.97 × 105 L/mol based on the experimental data. This KSV value is much higher than those in traditional organic compounds for sensing of Fe3+ (typical KSV of about 104 L/ mol).32 A linear standard calibration curve was obtained between ΔF = (F0 − F) and concentration of Fe3+ with correlation coefficient of R > 0.99 as presented in Figure S4. The limit of detection (LoD) calculated from this plot is 9.7 nmol/L (0.55 ppm). Moreover, we investigated the selectivity of S,N-CDs on the different ions. Figure 4c shows the fluorescence intensities of S,N-CDs with and without different metal ions (Li+, Na+, K+, Ag+, Zn2+, Pb2+, Ni2+, Mn2+, Fe2+, Cu2+, Co2+, Cd2+, Au3+, Cr3+, and Fe3+). It clearly shows that the fluorescence intensity almost remains unchanged (1−5% decrease) except for Cu2+ (∼30% decrease) and Fe3+ (>90% decrease). This is because there is nonspecific interaction between functional groups and metal ions.11 Figure S5 shows the antijamming capability of S,N-CDs and Fe3+-CDs in the presence of other metal ions. That implies the most that metal ions can coexist in the solution for the Fe3+ detection without any interference. The results suggest that the detection of S,NCDs on Fe3+ has high selectivity. We summarize the recent fluorescent carbon dots and graphene quantum dots for Fe3+ detection in Table S1. It should be noted that the S,N-CDs make distinction between Fe2+ and Fe3+ due to no FL
quenching in the presence of Fe2+. That is also employed for the FL recovering by addition the ascorbic acid (AA) because Fe3+ is reduced to Fe2+. So, Fe3+-S,N-CDs complexes can also be used as a sensor for the detection of AA via a turn on process. Figures 4d and S6 illustrate that the PL spectra and intensity difference of Fe3+-CDs increase with the concentration of AA. Based on the PL change vs the concentration of AA, we can calculate the LoD of AA detection is 44.3 nM (9.1 ppm), which is also highly sensitive for detection of AA. To understand the PL quenching process, The PL lifetime decays of S,N-CDs in the presence and absence of Fe3+ were investigated (Figure 4e). The average lifetime time of S,N-CDs is 12.9 ns and can be fitted into two components: 3.78 ns (ca. 37.7%) and 18.42 ns (ca. 62.3%). After addition with Fe3+ ions, the Fe3+-S,N-CDs lifetime decreased to 3.4 ns, of which decay time can also be fitted into two components: 3.55 ns (ca. 89.0%) and 2.64 ns (ca. 11.0%). The reduced lifetime indicates an ultrafast electron-transfer process in the Fe3+-S,N-CDs system and implying its dynamic quenching.41 The quenching rate (kq) is obtained from Ksv = kqτ0, where τ0= 12.9 ns. The calculated kq = 1.53 × 1013 L mol−1 s−1 is larger than the typical diffusion-controlled quenching rate (1010 M−1 s−1). According to Yang’s explanation, the high kq value may be on account of the combination of multiple Fe3+ ions with functional groups on the CDs (e.g., hydroxyl and amine) via coordinate bonds.11 Furthermore, we investigated functional groups of S,N-CDs on the effect of Fe3+ detection. Figure S7 shows the PL spectra of S,N-CDs-DMF prepared in DMF solution. The maximum emission peak locates at 500 nm, which is near green color. Figure S8 is the PL intensity of the mixture solution of S,NCDs-DMF and different metal ions. It clearly shows no PL quenching happens in the case of S,N-CDs-DMF, indicating that S,N-CDs-DMF has no detection function on the Fe3+. The FTIR spectrum (Figure S9) of S,N-CDs-DMF does not show the vibration peak at 2050 cm−1 either. From XPS analysis (Figure S10), no COOH group was found either.23 Those are the reasons why S,N-CDs-DMF cannot be used to detect Fe3+. 18552
DOI: 10.1021/acsami.7b04514 ACS Appl. Mater. Interfaces 2017, 9, 18549−18556
Research Article
ACS Applied Materials & Interfaces
Figure 5. Fluorescent images of tumor spheres of Huh7 cells (a) and Hep3B cells (b) incubated with S,N-CDs and S,N-CDs-Dox for 24 h imaged under white light and 488 and 550 nm excitation and merged images for 200× magnification. (c, d) Number of tumor spheres of Huh7 and Hep3B cells for culture with S,N-CDs and S,N-CD-Dox.
Figures 4f and S11 illustrate the change of fluorescence intensity of S,N-CDs and Fe3+-S,N-CDs with addition of ethylenediaminetetraacetic acid (EDTA), which is a common ligand with multiple carboxylic groups for Fe3+. The emission intensity of S,N-CDs dropped to 40% when Fe3+ was added into S,N-CDs solution. Subsequently, excess EDTA was added into the above Fe3+-S,N-CDs solution; it led to the emission intensity being recovered to ∼70%. When AA was added into the Fe3+-CDs solution, AA worked as a reductant as well and could reduce Fe3+ into Fe2+; Fe2+ only slightly quenched the fluorescence of S,N-CDs, and thus the emission was almost fully recovered. It indicated that EDTA only released -COOH from the binding between Fe3+ and -COOH on S,N-CDs. These results indicate that -COOH is part of the source of PL quenching in the Fe3+ detection. FTIR spectra (Figure S12) show that the -SCN characterized peak at 2050 cm−1 disappears and turns into double weak peaks at 2178 and 2017 cm−1, when Fe3+-S,N-CDs complexes formed. At the same time the 1704 cm−1 peaks disappear. These changes indicate that Fe3+ forms complexes with COOH and SCN groups on the S,N-CDs. EDTA was added into the e3+-S,N-CDs mixture due to strong coordination capability between Fe3+ and EDTA, which released part of Fe 3+ from COOH on S,N-CDs. The characterized peak at 1704 cm−1 appears again with the presence of EDTA. That results in part of PL being partly recovered. However, the peak at 2050 cm−1 does not show up with addition of EDTA indicating that -SCN group is not released. When AA was added into Fe3+-S,N-CDs, both peaks at 2050 and 1704 cm−1 are recovered, further confirming that both - SCN and -COOH are released and thus the PL emission is fully recovered. Bioimaging and Theranostic Agent. S,N-CDs in this study display excitation-dependent emission and the strongest PL emission at 600 nm under excitation light of 560 nm. The excellent property of continuous excitation-dependent full-color emission makes S,N-CDs applied in optical bioimaging. First, the relative viabilities of HepG2 cells exposed to S,N-CDs were measured using the CCK8 assays to evaluate the cytotoxicity of S,N-CDs.42All the samples exhibited a dose-dependent cytotoxicity. The S,N-CDs showed acceptable low cytotoxicity
(Figure S13) even after 72 h culture. Then, Huh7, Hep3B, and HepG2 cells are selected for the in vitro bioimaging. After culture with S,N-CDs for 24 h, most of the cells displayed green and red color under 488 and 550 nm irradiation (Figure 5a,b. S14), respectively. Compared with other fluorescent labeling agents, the property of multicolor emission of CDs makes it possible to freely choose the excitation wavelength in practical applications to prevent autofluorescence. Furthermore, a theranostic agent based on S,N-CDs and doxorubicin (Dox), which is a chemical therapy for tumor cells, was constructed by simply mixing two solutions together (Scheme S1). Dox can be loaded onto S,N-CDs by π−π stacking interaction due to both S,N-CDs and Dox having conjugated units. S,N-CDs/Dox complexes are dialyzed over 24 h in water to remove free small molecules. Figure S15 shows the UV−vis spectra of S,N-CDs, Dox and S,N-CDs/Dox. S,N-CDs/Dox exhibits adsorption bands of both S,N-CDs and Dox, indicating the composite form by simple stirring. Figure S16 exhibits PL spectra of S,NCDs/Dox under different excitation wavelengths. A new PL emission peak is observed besides the original emission from CDs at 595 nm. That further confirms the formation of complexes, and the PL of Dox has no effect on the emission of CDs. By calibration curve (Figure S16A), the Dox loading amount can be calculated as 0.28 mg/mg. Figure S16B shows the release of Dox in different pH environments. In neutral environment (pH = 7.4), the Dox keeps a very low concentration (
