Article pubs.acs.org/ac
Preparation of Yellow-Green-Emissive Carbon Dots and Their Application in Constructing a Fluorescent Turn-On Nanoprobe for Imaging of Selenol in Living Cells Qin Wang,†,‡ Shengrui Zhang,†,‡ Yaogang Zhong,§ Xiao-Feng Yang,*,† Zheng Li,§ and Hua Li*,†,∥ †
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi’an, Shaanxi 710127, P. R. China ‡ School of Chemistry and Environment Science, Shaanxi Sci-Tech University, Hanzhong, Shaanxi 723000, P. R. China § College of Life Sciences, Northwest University, Xi’an, Shaanxi 710069, P. R. China ∥ College of Chemistry and Chemical Engineering, Xi’an Shiyou University, Xi’an, Shaanxi 710065, P. R. China S Supporting Information *
ABSTRACT: Selenocysteine (Sec) carries out the majority of the functions of the various Se-containing species in vivo. Thus, it is of great importance to develop sensitive and selective assays to detect Sec. Herein, a carbon-dot-based fluorescent turn-on probe for highly selective detection of selenol in living cells is presented. The highly photoluminescent carbon dots that emit yellow-green fluorescence (Y-G-CDs; λmax = 520 nm in water) were prepared by using m-aminophenol as carbon precursor through a facile solvothermal method. The surface of Y-G-CDs was then covalently functionalized with 2,4-dinitrobenzenesulfonyl chloride (DNS-Cl) to afford the 2,4-dinitrobenzenefunctionalized CDs (CD-DNS) as a nanoprobe for selenol. CDDNS is almost nonfluorescent. However, upon treating with Sec, the DNS moiety of CD-DNS can be readily cleaved by selenolate through a nucleophilic substitution process, resulting in the formation of highly fluorescent Y-G-CDs and hence leads to a dramatic increase in fluorescence intensity. The proposed nanoprobe exhibits high sensitivity and selectivity toward Sec over biothiols and other biological species. A preliminary study shows that CD-DNS can function as a useful tool for fluorescence imaging of exogenous and endogenous selenol in living cells.
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probes to sensitively and selectively monitor Sec in living cells. To detect Sec selectively, the key is to differentiate Sec from other biological nucleophiles, especially biothiols such as cysteine (Cys) and glutathione (GSH), which are usually present in high concentrations in biological systems. Sec is a Cys analogue with a selenium-containing selenol group in place of the sulfur-containing thiol group in Cys.3 The pKa value of selenol in Sec is 5.24,4 which is significantly lower than that of its analogous biothiols (8.44 for Cys, 8.6 for GSH).5,6 As a consequence, Sec is normally present as nucleophilic selenolate (R-Se−) at physiological pH, whereas biothiols are mainly present as a less reactive neutral form (R-SH).7 Moreover, selenolate is more nucleophilic than its analogous thiolate.8 The above features open a window for selective detection of Sec over biothiols. On the basis of these properties, Maeda et al. reported the first fluorescent probe 3′-(2,4-dinitrobenzenesulfonyl)-2′,7′-dimethylfluorescein (BESThio) to discriminate Sec
elenium (Se) is a biologically essential micronutrient element and plays a significant role in several biological processes, such as cellular redox balance, immune responses, cancer prevention, and inflammation protection. Many different metabolites of Se, such as hydrogen selenide, selenocysteine (Sec), selenite, seleno-phosphate, selenodiglutathione, and charged Sec-tRNA, are synthesized in animals in the course of converting inorganic Se to organic forms and vice versa.1,2 Among them, Sec holds a particularly prominent position because it is an essential building block for selenoproteins (SePs), which are involved in various cellular functions and linked to several human diseases, such as cancers, cardiovascular diseases, neurodegenerative diseases, and inflammation. As Sec carries out the majority of the function of the various Secontaining species in vivo, there is a strong desire to develop reliable and rapid assays for Sec in biological systems. Fluorescence-based methods are powerful tools due to their high specificity and sensitivity and can be applied in living systems with high temporal/spatial resolution capability. Therefore, in order to investigate the essential roles of Sec in physiological functions, it is necessary to construct fluorescent © 2016 American Chemical Society
Received: October 10, 2016 Accepted: December 23, 2016 Published: December 23, 2016 1734
DOI: 10.1021/acs.analchem.6b03983 Anal. Chem. 2017, 89, 1734−1741
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Analytical Chemistry from its counterpart Cys.9 However, due to the intrinsic higher reactivity of the 2,4-dinitrobenzenesulfonate-caged compounds, BESThio can discriminate Sec from Cys only under acidic conditions (pH 5.8), which are not compatible with the neutral biological surroundings (pH ∼ 7.4) and thus prevent their application in living systems. Later, this issue was addressed by replacing the sulfonate connection with the corresponding sulfonamide linkage to develop 2,4-dinitrobenzenesulfonamidebased fluorescent probes for Sec.10 The resulting probes exhibit improved selectivity toward Sec over other biothiols under physiological pH conditions. In an advance, several other groups have adopted the same strategy to develop Sec probes with improved properties, such as NIR emission11 and higher sensitivity.12 However, the current reported selenol fluorescent probes are all based on organic fluorophores, which generally show poor photostability and thus make long-term imaging difficult. Therefore, searching for better fluorophores has been a constant and critical effort for bioimaging studies. Fluorescent carbon dots (CDs), a new type of carbon-based nanomaterials, have gained tremendous attention in recent years because of their unique optical properties, high photostability, biocompatibility, and lower toxicity as compared to traditional fluorescent dyes.13,14 These features make CDs a particularly useful tool for bioimaging and biosensing. In recent years, CD-based probes for a variety of analytes have been developed.15−25 However, the excitation wavelength of these reported probes is mainly located in the blue-light region, which hinders their applications for imaging studies in living cells. Therefore, exploration of CD-based fluorescent probes suitable for monitoring targeting species in living cells or biological processes still remains at an early stage. In this work, we developed a long-wavelength fluorescent turn-on nanoprobe for Sec by surface functionalization of CDs with a highly specific Sec recognition element. In the proposed nanoprobe, yellow-green-emissive CDs (Y-G-CDs) prepared from m-aminophenol (mAP) were selected as the fluorescent reporter, and the 2,4-dinitrobenzenesulfonyl (DNS) group was selected as the recognition unit and covalently coupled onto the surface of Y-G-CDs via sulfonamide bond to afford the 2,4dinitrobenzene-functionalized CDs (CD-DNS) as the desired probe. CD-DNS is almost nonfluorescent. However, upon incubation with Sec in aqueous solution at room temperature, the DNS moiety of CD-DNS can be readily cleaved by selenolate through a nucleophilic substitution process, resulting in the formation of highly fluorescent Y-G-CDs and hence leads to a dramatic increase in fluorescence intensity (Scheme 1). On the basis of the above mechanism, a CD-based fluorogenic
probe (CD-DNS) for Sec in aqueous solution was developed. The probe shows excellent sensitivity and specificity toward Sec over biological thiols. Preliminary studies show that CD-DNS has less toxicity and can be applicable for fluorescence imaging of exogenous and endogenous selenol in living cells.
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EXPERIMENTAL SECTION Materials and Instruments. 2,4-Dinitrobenzenesulfonyl chloride (DNS-Cl) was obtained from Sigma-Aldrich. LSelenocystine was purchased from J&K Chemicals Company. Protein tyrosine phosphatase 1B (PTP1B) was purchased from Sino-Biological (Beijing, China). mAP, sodium selenite (Na 2 SeO 3 ), Cys, GSH, tris(2-carboxyethyl) phosphine (TCEP), homocysteine (Hcy), p-toluenesulfonyl chloride, glucose, and other amino acids were all purchased from Aladdin Ltd. (Shanghai, China). All other reagents were of analytical grade and purchased from commercial supplies. Distilled water was used throughout this study. Transmission electron microscopy (TEM) and high resolution TEM (HRTEM) images were performed on an electronic microscopy (GZF 2.0, FEI Electron optics). X-ray powder diffraction (XRD) patterns were obtained using an Xray diffractometer (Bruker, Germany) with Cu Kα radiation (λ = 1.5178 Å). The 2θ scanning range was from 10° to 70° with a scanning speed of 0.1°/s. Fourier transform infrared (FT-IR) spectra were obtained on a Vertex 70 FT-IR spectrometer (Bruker, Germany), using a diamond ATR attachment. X-ray photoelectron spectroscopy (XPS) was carried out with ESCALAB 250Xi (Thermo Scientific). The fluorescence spectra and relative fluorescence intensity were measured with a PerkinElmer LS-55 fluorescence spectrometer. Absorption spectra were recorded using a Shimadzu UV-2550 spectrophotometer. The pH measurements were carried out on a Sartorius PB-10 pH meter. Fluorescence imaging was performed by an Olympus FV1000 confocal laser scanning microscope. Preparation of CDs. To mAP (0.1 g) in C2H5OH (10 mL) was added concentrated hydrochloric acid (15 μL) and nitric acid (5 μL), and then, the solution was transferred into a 25 mL poly(tetrafluoroethylene)-lined stainless steel autoclave. After heating at 180 °C in an oven for 12 h and cooling down to room temperature, a red-brown suspension was obtained. The crude product was then purified with a silica column chromatography using CH2Cl2/CH3OH (5:1, v/v) as eluents. After evaporating the solvent and further drying under vacuum, seven purified CD samples with different fluorescence were finally obtained (Scheme 2). Preparation of CD-DNS. To a solution of Y-G-CDs (20 mg) and Et3N (100 μL) in a mixture of anhydrous CH2Cl2 (30 mL) and DMF (2 mL), DNS-Cl (200 mg, mixed with 6 mL of anhydrous CH2Cl2) was added dropwise. The mixture was stirred at room temperature for 2 h. After that, the solution was washed with water, dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The crude residue was further purified by silica gel flash column chromatography using CH2Cl2/CH3OH (10:1, v/v) as eluents to afford CD-DNS as a dark brown solid. Preparation of p-Toluene-Functionalized CDs (CDPTS). CD-PTS was prepared by treating Y-G-CDs with ptoluenesulfonyl chloride using the same procedure described for the preparation of CD-DNS, and its structure (shown in Figure S1) was confirmed by an FT-IR spectrum (Figure S2).
Scheme 1. Schematic Illustration of the Sensing Mechanism of CD-DNS for Seca
a
Other function groups on CDs are omitted for clarity. 1735
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Analytical Chemistry Scheme 2. Synthesis Route for Y-G-CDs and CD-DNSa
a
Other function groups on CDs are omitted for clarity.
Cell Cultures and Fluorescence Imaging. L929 cells were seeded in culture plates containing sterile coverslips and were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin (100 U mL−1), and streptomycin (100 μg mL−1) at 37 °C in a humidity atmosphere under 5% CO2 for 24 h. The medium was removed, and the adherent cells were washed with phosphate buffer saline (PBS) (pH 7.4) three times. Then, the cells were treated with the CD-DNS (20 μg mL−1) for 12 h. After washing with PBS three times to remove the remaining probe, the cells were further incubated with Sec (40 μM) for 30 min at 37 °C. After washing the cells with PBS three times, cell imaging was then immediately performed by an Olympus FV1000 confocal laser scanning microscope. The excitation wavelength for CDDNS was at 458 nm; emission collection was at 475−575 nm. To induce the endogenous Sec, L929 cells were exposed to Na2SeO3 (5 or 10 μM) for 12 h, and then, the cells were further incubated with the CD-DNS (20 μg mL−1) for 6 h at 37 °C. After that, the staining solution was replaced with fresh PBS to remove the remaining free probe. Cell imaging was then performed using the conditions described above.
Figure 1. Photographs of seven CD samples in aqueous solution under visible (A) and 365 nm UV light (B). (C) Corresponding fluorescence spectra of the seven samples with the maximal emission wavelength at 450, 515, 520, 534, 550, 575, and 611 nm, respectively. Each spectrum was obtained under the maximal excitation wavelength of the sample.
thus are not suitable for further construction of fluorescent nanoprobes. The maximal emission wavelengths of G-CDs and Y-G-CDs in aqueous solution are 515 and 520 nm, respectively (Figure 1C). Their QYs in water were determined to be 27.7% and 28.6% by using fluorescein (QY = 95% in 0.1 M NaOH) as a fluorescence standard,33 respectively. The resulting G-CDs and Y-G-CDs in aqueous solution exhibited a long-term homogeneous phase without any noticeable precipitation at room temperature, and as shown in Figure S4, no obvious fluorescence intensity changes were observed even after 36 h. Furthermore, the pH-dependent fluorescence behaviors of G-CDs and Y-G-CDs in aqueous solution were investigated. As shown in Figure S5, the fluorescence intensity of G-CDs gradually increased with pH decreasing from 9.18 to 4.92. Obviously, acidic conditions are favorable for their fluorescence measurement. In the case of YG-CDs, however, a strong fluorescence intensity of Y-G-CDs was obtained in the pH range of 7.17−8.67. Apparently, Y-GCDs are ideal for bioimaging studies at the physiological pH conditions (pH ∼ 7.4). Thus, Y-G-CDs were selected as the fluorophore to construct nanoprobe for Sec in living cells. Structure, Composition, and Optical Properties of YG-CDs. With Y-G-CDs in hand, the TEM and HRTEM images of Y-G-CDs were first performed. As shown in Figure 2A, Y-GCDs were spherical in shape and well dispersed in water, and the sizes of Y-G-CDs were mostly distributed in the range from 1.8 to 4.3 nm, with an average size of 2.8 nm (Figure 2B). The
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RESULTS AND DISCUSSION Design and Synthesis of Y-G-CDs. To construct CDbased probes with cellular imaging capability, there is a prerequisite to synthesize CDs presenting high photoluminescence quantum yields (QYs) and long excitation wavelength. Recently, CDs prepared by using phenylenediamines as precursors attract our attention due to their excellent fluorescence properties such as high QYs and long excitation/ emission wavelengths.26−32 Encouraged by these results, we assumed that CDs with high QYs as well as long-wavelength emission behavior could be prepared by using their analogous precursors. Here, mAP was chosen as the carbon source to prepare CDs via a facile one-pot solvothermal method under acidic conditions (Scheme 2). The as-prepared products were actually mixtures of CD samples with different emission behaviors (Figure S3), which could be separated via flash column chromatography. As shown in Figure 1, seven CD samples were obtained from the above reaction mixture, and they exhibit distinct fluorescence characteristics. Among these samples, the green-emissive CDs (G-CDs) and Y-G-CDs could be finally obtained in 6.7 and 18.3 wt % yields, respectively, while the yields of other five CD samples were very few and 1736
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Analytical Chemistry
C−O−C/N−O, and −OH;26,40,41 The Cl 2p3 band of Cl element contained two peaks, representing benzoquinone and benzene Cl,42,43 which may derive from hydrochloric acid added in the reaction mixture. These XPS assignments were further verified by FT-IR characterization. As shown in Figure 3B, the FT-IR spectrum shows that Y-G-CDs possess −OH (3575 cm−1), −NH2 (3021 cm−1), aliphatic CH2 (2834 cm−1), OC−O− (1820 cm−1), CO (1612 cm−1), CC (1446 cm−1), C−O−C (1358 cm−1), and C−NH (1241 cm−1) functional groups or chemical bonds.44−46 The results obtained from XPS and FT-IR are consistent, indicating that −OH and −NH2 groups are present on the surface of Y-G-CDs. The absorption and fluorescence spectra of Y-G-CDs in aqueous solution were shown in Figure 4. In the UV region of
Figure 2. TEM and HRTEM (inset) images (A), histograms of the particle size distribution (B), and XRD pattern (C) of Y-G-CDs.
HRTEM images showed that Y-G-CDs exhibited identically well-resolved lattice fringes with interplanar spacings of 0.21 nm, corresponding to the (100) in-plane lattice of graphene (inset of Figure 2A).34 The XRD pattern of Y-G-CDs (Figure 2C) exhibited a broad diffraction peak centered at about 25°, which further confirms the graphene structure of the Y-GCDs.35 Subsequently, Y-G-CDs were characterized by XPS and FTIR to identify their surface composition and chemical state. The XPS results demonstrated that Y-G-CDs mainly contained four elements: C, N, O, and Cl (Figure 3A). In the high-resolution spectra (Figure 3C−F), the C 1s band could be deconvoluted into three peaks, corresponding to C−C/CC, C−N/C−O, and OC−O.27,36 The N 1s band showed distinctive peaks, representing pyridinic N, amino N, and Nitro N.37−39 The O 1s band exhibited characteristic peaks, representing CO/Si−O,
Figure 4. Absorption (Abs) and fluorescence emission spectra (Em) of Y-G-CDs under different excitation wavelengths.
the absorption spectrum, a single peak was observed at 264 nm for Y-G-CDs, corresponding to the π−π* transitions of the conjugated CC units from the carbon core, leading to weak observed fluorescence emission.35,47 In the lower-energy region, a broad absorption band centered at 442 nm was observed, which was assigned to the n−π* transition from the surface/ molecular section, resulting in strong fluorescence emission.27,48 Further, the fluorescence spectra of Y-G-CDs in aqueous solution were recorded. Unlike most reported CDs,49 the fluorescence spectra of Y-G-CDs were excitationindependent when the excitation wavelength was progressively increased from 300 to 470 nm, indicating that the emission properties of Y-G-CDs are mostly dependent on the surface state.44 The maximal excitation and emission wavelengths of YG-CD in water are 450 and 520 nm, respectively (Figures 4 and S6). In addition, the maximal excitation wavelength of Y-G-CD is close to its absorption band in the lower energy region, confirming that absorption structures and luminescent centers of Y-G-CDs are identical. Apart from its superior optical properties, Y-G-CDs are proved to contain many amino groups on their surface, thus imparting them the suitability for subsequent functionalization with recognition units. Hence, Y-G-CDs were selected as a fluorophore to construct nanoprobe for Sec in living cells. We envisioned that the well-elucidated mechanism for sensing Sec via selective cleavage of 2,4-dinitrobenzenesulfonamide functionality might be incorporated into Y-G-CDs to afford a CDbased nanoprobe for Sec. Therefore, it occurred to us to develop CD-DNS as a fluorescent turn-on nanoprobe for selenol. Our rationale is depicted in Scheme 1.
Figure 3. Characterization of the structure and functional groups of YG-CDs. (A) XPS survey; (B) FT-IR characterization; high-resolution XPS survey scan of (C) C 1s, (D) N 1s, (E) O 1s, and (F) Cl 2p3 spectra of Y-G-CDs. Each band was deconvoluted following the literature.26,27,36−43 1737
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Analytical Chemistry CD-DNS was synthesized by conjugating the strongly electron-withdrawing DNS group on the surface of Y-G-CDs via a sulfonamide bond (Scheme 2). Then, the element composition of CD-DNS was characterized by XPS and FT-IR. As shown in Figure S7A, CD-DNS contained a new element, S 2p (164 eV), and in the high-resolution spectra, XPS data analysis of the N 1s spectra indicated a dramatic increase in the content of Nitro N (Figure S7C,D and Table S1). The results were further confirmed by FT-IR measurements, and the absorption peaks of −NO2 (1539 and 1345 cm−1) and −SO2− (1386 and 1193 cm−1) were observed in the FT-IR spectrum of CD-DNS (Figure S7B). The above results provide strong evidence that DNS was successfully attached on the surface of Y-G-CDs. With the probe in hand, the absorption and fluorescence spectra of CD-DNS were first investigated. As seen from Figure S8A, the absorption maximum of CD-DNS was located at 355 nm, which was blue-shifted about 87 nm in comparison with that of Y-G-CDs. Fluorescence studies revealed that CD-DNS itself is almost nonfluorescent (Figure S8B). We hypothesized the plausible mechanism for the quenching of the fluorescence of Y-G-CDs is the intramolecular photoinduced electron (PET) process from the excited fluorophore to the electron-deficient 2,4-dinitrobenzyl moiety.50,51 To prove the involvement of the PET process, CD-PTS was prepared as a control. In this case, PET from Y-G-CDs to the benzyl group should be disfavored because the strong electron-withdrawing effect of the nitro groups is removed. The fluorescence spectra of Y-G-CDs, CDDNS, and CD-PTS were recorded, and the results are shown in Figure S8B. It can be observed that CD-PTS gives stronger fluorescence emission than that of CD-DNS, while its emission intensity is significantly lower than that of Y-G-CDs. The above results provide strong evidence that the nitro groups are responsible for the fluorescence quenching of Y-G-CDs via an intramolecular PET process. On the other hand, the surface modification also contributes to the fluorescence quenching, as it changes the surface state of Y-G-CDs. Obviously, the abovementioned two factors are both involved in the fluorescence quenching of Y-G-CDs, and the latter contributes to a larger extent than the former. Next, the capability of CD-DNS to detect selenol was examined by introducing Sec to the solution of CD-DNS in phosphate buffer (20 mM, pH 7.4). As Sec is not stable in aqueous solution, it was generated in situ by mixing TCEP and selenocystine.52 As shown in Figure 5, the free probe is almost nonfluorescent. However, upon progressive addition of Sec (0− 40 μM), the fluorescence intensity of CD-DNS solution at 514 nm gradually increased until it reached a plateau after the addition of 40 μM of Sec. The fluorescence intensity at 514 nm increased linearly with Sec concentration in the range of 0.2− 30 μM. The detection limit was calculated to be 23 nM (3σ). These results demonstrate that CD-DNS can detect Sec with high sensitivity. The absorption spectral changes of CD-DNS upon addition of Sec were recorded. As shown in Figure S9, the free CD-DNS shows a broad absorption peak centered at 355 nm. Upon treating with Sec, a new absorption band centered at 442 nm emerged, which agreed well with that of parent Y-GCDs, indicating the restoration of Y-G-CDs in the above sensing process. It was also observed that the absorption band at 355 nm increased significantly in the above reaction solution, which is apparently contributed by the 2,4-dinitrophenyl selenoether (1) generated in the reaction mixture (shown in
Figure 5. (A) Fluorescence spectra (λex = 450 nm) of CD-DNS (20 μg mL−1) upon addition of increasing concentrations of Sec (0−40 μM) in phosphate buffer (20 mM, pH 7.4, containing 2% acetone) after 12 min of incubation. Sec was generated in situ by the treatment of selenocystine with the reducing agent TCEP (120 μM). Inset: photographs of CD-DNS alone and in the presence of Sec exposed to a UV lamp at 365 nm. (B) Linear relationship between the fluorescence intensity and the concentrations of Sec (0−30 μM). λex/λem = 450/514 nm.
Scheme 1). The above results further confirm the nuleophilic aromatic substitution between CD-DNS and Sec. The kinetic behavior of CD-DNS toward Sec was initially examined (Figure 6). The free probe exhibited almost no
Figure 6. Time course of fluorescence intensity of CD-DNS (20 μg mL−1) in the presence of Sec (40 μM), Cys (100 μM), and GSH (1 mM) in phosphate buffer (20 mM, pH 7.4, containing 2% acetone). λex/λem = 450/514 nm. 1738
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Analytical Chemistry changes in fluorescence intensity. With the introduction of Sec to the solution of CD-DNS, however, it can be seen that the fluorescence intensity of the reaction mixture was increasing with time and reached a plateau at 12 min. In the case of GSH (1 mM) and Cys (100 μM), although biologically relevant concentrations of them exhibited a slight increase in emission intensity, it induced no significant effect toward the sensing of Sec in biological systems. Furthermore, the effect of pH on the fluorescence responses of CD-DNS toward Sec (40 μM) and GSH (1 mM) was investigated. As shown in Figure 7, the fluorescence intensity of
Figure 8. Fluorescence intensity of CD-DNS (20 μg mL−1) in the presence of various analytes in phosphate buffer (20 mM, pH 7.4, containing 2% acetone) for 12 min. Sec, 40 μM; PTP1B, 2.0 μg mL−1; Cys and Hcy, 100 μM. The concentrations of other analytes are 1.0 mM. λex/λem = 450/514 nm. Data were expressed as mean ± SD of three experiments.
enhancement under identical conditions. Furthermore, the fluorescence response of the probe to Sec in the presence of different competing species was examined. As shown in Figure S10, most of coexisting species displayed minimum interference in the detection of Sec. These data demonstrate that the probe has a high selectivity toward Sec over other biologically relevant species. This selectivity is due to not only the relatively lower pKa (SeH) of Sec but also Sec behaving as a stronger nucleophile than biothiols.9 It was previously reported that the character of linkage between the fluorophore and 2,4-dinitrobenzene moiety determines the probes’ selectivity. The sulfonate connection failed to discriminate the Sec from biothiols under physiological pH of 7.4, while probes with sulfonamide linkages exhibit promising selectivity.10 Therefore, the high selectivity of CDDNS toward Sec over biothiols indicates that the sulfonamide connection is predominantly present at the surface of CD-DNS. However, the formation of 2,4-dinitrobenzenesulfonate between the hydroxyl groups on the surface of Y-G-CDs and DNS-Cl cannot be completely excluded because a slight increase in fluorescence intensity was observable when CDDNS was treated with a large amount of biothiols under physiological conditions. Exogenous and Endogenous Sec Imaging in Living Cells. Encouraged by the above-mentioned outcome, some experiments were performed to evaluate CD-DNS in living cell imaging assays using L929 cells as a model cell line. Initially, the cytotoxicity of CD-DNS was evaluated using a standard MTT assay. The results showed that CD-DNS poses negligible cytotoxicity to L929 cells below 50 μg mL−1 (Figure S11). As the physiological concentration of Sec is low in cells, we first determined if CD-DNS could respond to the exogenous Sec. After treating L929 cells with Sec, a bright yellow-green fluorescence signal appeared (Figure 9B), while the control cells exhibited dim fluorescence (Figure 9A), indicating that the cellular thiols do not interfere with the detection of Sec by CDDNS. This exciting observation motivates us to further determine the endogenous Sec stimulated by Na2SeO3 in living cells. As a precursor of Sec biosynthesis, Na2SeO3 itself did not react with CD-DNS, and the supplement of cells with Na2SeO3 could significantly increase Sec levels in cells.53,54 We further treated the cells with Na2SeO3 (two different
Figure 7. Fluorescence intensity changes of CD-DNS (20 μg mL−1) in the absence and presence of Sec (40 μM) or GSH (1 mM) in phosphate buffer with different pH values. λex/λem = 450/514 nm.
CD-DNS did not show significant changes in the pH range of 4.92−9.18, indicating that this probe is stable in a wide pH range. Upon introducing Sec to the solution of CD-DNS, a strong fluorescence signal was obtained in the pH range of 7.4− 9.18, which meets the requirement for Sec sensing in neutral biological systems. These results can be explained by the fact that higher pH facilitates the ionization of Sec to selenolate anion and hence promotes the nucleophilic reaction to release the emissive Y-G-CDs. As the pH value further increases from 8.6 to 9.18, the emission intensity decreased slightly. This is apparently due to the pH-dependent fluorescence behavior of Y-G-CDs. In the case of GSH, it gives a weak fluorescence signal when the pH value is
