One-step synthesis of lable-free ratiometric fluorescence carbon dots

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Functional Nanostructured Materials (including low-D carbon)

One-step synthesis of lable-free ratiometric fluorescence carbon dots for the detection of silver ion and glutathione and cellular imaging application Yuan Jiao, Yifang Gao, Yating Meng, Wenjing Lu, Yang Liu, Hui Han, Shaomin Shuang, Lei Li, and Chuan Dong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b01319 • Publication Date (Web): 12 Apr 2019 Downloaded from http://pubs.acs.org on April 12, 2019

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ACS Applied Materials & Interfaces

One-step synthesis of lable-free ratiometric fluorescence carbon dots for the detection of silver ion and glutathione and cellular imaging application Yuan Jiao, a Yifang Gao, a Yating Meng, a Wenjing Lu, a Yang Liu, a Hui Han, a Shaomin Shuang, a Lei Li, b Chuan Dong a* Institute of Environmental Science, and School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006,China b Department of Chemical & Petroleum Engineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA, 15261, USA KEYWORDS: Long-wavelength emission; Carbon dots; Ratiometric fluorescent; Silver ion; Glutathione; Bioimaging a

ABSTRACT: The construction of ratiometric fluorescence assay have displayed fantastic advantages in improving semi-quantitative visualization capability by presenting successive color changes. Herein, long-wavelength emission nitrogen-doped carbon dots (NCDs) were developed for intrinsic ratiometric detection of silver ion (Ag+) and glutathione (GSH) accompanying with visualization fluorescence variation of orange and green. The lable-free N-CDs were favorably obtained through one step hydrothermal synthesis and displayed single long-wavelength emission at 618 nm under the excitation wavelength of 478 nm. Interestingly, a ratio rising peak emerges at 532 nm along with the emission at 618 nm decreases with the introduction of Ag+, which exhibit ratiometric fluorescence emission characteristics (I618 nm/I532 nm) in the range of 0-140 μM with significant fluorescence various from orange to green. Furthermore, the fluorescence of CDs@Ag(I) can be effectively ratiometric recovered by virtue of a specific reaction of GSH with Ag+ accompanying with the fluorescent of the solution returns from green to orange. In addition, the N-CDs hold excellent biocompatibility which can implemented as visualization biosensing platform for intracellular determination of Ag+ and GSH, demonstrating that proposed N-CDs has tremendous potential in biological systems.

INTRODUCTION As a representative heavy metal ions, silver has drawn considerable attention recently as a result of its widespread application in photograph1, jewelry2, electronic equipment3 and pharmacy4. The excess accumulation of silver ions (Ag+) may has massive influence on human health5 and further results in various diseases such as exhaustion of organ function6, growth retardation7 and mitochondrial damage8 through the promotion of oxidative stress. On the other hand, glutathione (GSH), an essential biothiol, play a critical role in the maintenance of reversible redox reactions9-10, cellular signal transduction11, detoxification and metabolism12 etc. The abnormal levels of GSH are closely associated with several diseases such as diabetes, cancer, and human immunodeficiency virus13-15. The analysis of GSH levels in human plasma or urine becomes extremely critical for the diagnosis of early disease16-17. Notably, GSH is a strong Ag+ binder, which can capture silver ions and make it escape from the sensor18. Based on this consideration, the development of relatively cost-effective, selective and sensitive methods for determination trace amount of Ag+ and GSH in biological system are of important concern. So far, numerous alternative approaches have been established for detection of Ag+ and GSH, increasing attentions have focused on fluorescence-based sensors due to its green

synthesis procedures, superior sensitivity and outstanding optical feature19-21. Among them, ratiometric fluorescence assay have displayed fantastic advantages of improving sensitivity at trace quantity levels of analyte and eliminate environmental effects, as well as improve semi-quantitative visualization capability by presenting successive color changes22-24. Such ratiometric fluorescence assay is frequently composed of double individual fluorophores with distinguishably fluorescence emission wavelengths, in which measuring the ratio of two fluorescent peaks as the sensing signal instead of the absolute intensity of one peak25. Up to now, numerous advanced fluorescent materials such as gold nanoparticles26-28, silver nanoparticles29-30 organic small molecules31-33 and semiconductor quantum dots34-35 etc. have been essentially utilized for the construction of ratiometric fluorescence probes. However, most of these fluorescent sensors possess larger particle sizes and expensive or hard to prepare etc. Therefore, the development of low-cost and non-toxic ratiometric fluorescence sensors with excellent optical properties becomes extremely critical to biological system. As a class of photoluminescence (PL) nano-materials, carbon quantum dots (CDs) have been employed favorably in drug delivery36, biological labeling37, fluorescent sensor38 as a result of their unique optical properties, low toxicity and advantageous biocompatibility39-40. In most of applications,

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CDs-based ratiometric nanosensors were generally designed by self-assembling or covalently conjugation with another fluorophores. For instance, Yan et al.41 constructed a nanohybrid ratiometric fluorescence nanosensor by comprising blue-emissive CDs with red-emissive GSH-CdTe QDs and demonstrated its potential application for visual detection of NO2. Yu et al.42 designed a FRET assembly based on the noncovalent interaction between CDs and AuNCs for ratiometric detection of cysteine. Zou et al.43 developed a ratiometric fluorescence nanohybrid composite by coating QDs-loaded silica NPs with CDs on the outer surface via a carbodiimidemediated approach for detection of Cu2+. Ma et al.44 reported a facile strategy by compositing CDs and Ru(bpy)32+ to construct a ratiometric sensing platform for specificity detection of ClO− and demonstrated their utilization in bioimaging. All these inspiring research opened a new route for the construction of CDs-based ratiometric fluorescent probes. However, these nanosensors are typically composed of two separate materials with different fluorescence emission wavelengths, which requires complicated separation and purification operate in preparation process and further results in time-consuming and cumbersome procedures. Very recently, some other researchers45-47 reports that one-step synthesis of ratiometric fluorescent CDs without further modified and demonstrated their fluorescent-based biosensing applications. Most of such strategy exhibit short-wavelength emission, which one of the fluorescence acts as a reference signal with negligible change in fluorescence intensity and another fluorescence is used as a detection signal45. Hence, the development long-wavelength emission carbon dots and further construct CDs-based ratiometric fluorescence nanosensors for monitoring of Ag+ and GSH in biological system are still of important concern. In this work, we fabricated a label free ratiometric fluorescence N-CDs via one step hydrothermal strategy with neutral red and triethylamine as precursors. The produced water-solubility N-CDs exhibits orange fluorescence with emission wavelength at 618 nm. The N-CDs displayed intrinsic ratiometric fluorescence variation (I618 nm/I532 nm) against the introduction of Ag+ upon one excitation wavelength of 478 nm, which attributed to the N - Ag ligand-to-metal charge transfer. With the addition of GSH, the fluorescence of the CDs@Ag(I)

system was recovered from green back to orange along with the ratio of I618 nm with I532 nm is increases the same as the initial value. The fluorescent N-CDs also holding some other exceptional characteristics such as admirable reversibility, outstanding stability, favorable biocompatibility and brilliant cellular labeling capability. Taking advantage of these exclusive properties, the N-CDs was employed as a kind of ratiometric fluorescence biosensors to noninvasively monitor stimulus-responsive changes of intracellular Ag+ and GSH in living cells. The produced N-CDs were demonstrated as a promising nanomaterials for construction of dual sensing platform based on fluorescence ratiometric dynamic for sensitive sensing of Ag+ and GSH, which provided a new insight in clinical diagnosis and other biomedical fields (See Scheme 1). EXPERIMENTAL SECTION Materials. Neutral red (NR) and triethylamine were purchased through Aladdin Ltd (Shanghai, China). Phosphate buffer saline (PBS) solutions at different pH were arranged by titrating 0.02 M phosphate solution with NaOH (1.0 M) and H3PO4 (1.0 M). Various metal ion solutions with a concentration of 0.1M were formulated from chloride and nitrat salts of K+, Ca2+, Na+, Mg2+, Al3+, Zn2+, Co2+, Cr6+, Fe3+, Cu2+, Hg2+, Cr3+, Mn2+, Pb2+, Ni2+, Ba2+, Fe2+, Cd2+, Sn2+, Ag+. The solutions of different amino acid (Phe, Lys, Thr, Hcy, Cys, Asp, Tyr, Ser, Ile, Glu, Gln, Leu, His, Gly, Trp, Val, Pro, Arg, Ala, Met, GSH) were prepared in the concentration of 0.01M. The ultrapure water ( ≥ 18.25 MΩ.cm) was applied in the all experiment. Preparation of N-CDs. The orange emission N-CDs were obtained via one-step strategy. 0.0059g of neutral red was firstly dissolved with 20 mL ultrapure water, then introducing 300 μL of triethylamine into above system for the cross-linking of carbon materials. The obtained uniform solution was sealed in a Teflon autoclave and heated at 200 ℃ for 5 h. After that, the suspensions was centrifuged at 10,000 rpm for 15 min and then further filtered by a 0.22 μm pore diameter microporous membrane. The purified solution was freeze-dried to obtain solid state N-CDs. Detection of Ag+ and GSH. The fluorescence detection experiment were performed in 0.02 M phosphate buffer saline (pH=7.4) with the concentration of 0.1 mg∙mL-1. Firstly, 200 μL of N-CDs aqueous solution (1.0 mg∙mL-1) was added into 2.0 mL of PBS at pH=7.4. Then different concentrations of Ag+ were introduced separately and the fluorescence emission spectra were recorded. For the detection of GSH, the fluorescence spectrum were collected by adding different concentrations of GSH into the CDs@Ag(I) (140 μM) system. The selectivity of the N-CDs toward Ag+ and GSH were demonstrated by adding various metal ions and amino acids take the place of Ag+ and GSH at the same conditions as above mentioned. All measurements were conducted in triplicate.

Scheme.1 Schematic diagram of ratiometric nanosensors for the detection of Ag+ and GSH and ratio bioimaging application.


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ACS Applied Materials & Interfaces Cellular Imaging. Human hepatic carcinoma SMMC7721 cells were cultured in culture medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a 5.0% CO2 incubator at 37 ℃for 48 h. The washed SMMC7721 cells were incubated with a typical thiol blocker N-ethylmaleimide (NEM, 1.0 mM) for 30 minutes. Subsequently, introducing 0.3 mg∙mL-1 N-CDs aqueous solution for an additional 2h at 37℃and then washing the cells three times with PBS buffer solution (pH=7.4). Immediately, fluorescence images were collected on a confocal laser scanning microscope. 25 μL of 0.01M Ag+ was introduced into the living cells system firstly. Following, 25 μL of GSH with a concentration of 0.01M was added in the sample. Intracellular fluorescence images were collected on a LSCM. RESULTS AND DISCUSSION Characterizations of N-CDs. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) were expansively utilized as influential instruments in the study of morphology of N-CDs. As described in Fig.1a, the N-CDs are nearly spherical with an average diameter of 2.78 ± 0.23 nm. The full width at half maximum of the Gaussian fitting curve is 0.6 nm, which certificates that the N-CDs have uniform dimension. Further confirmed by AFM (Fig.1b), N-CDs exhibit particle heights of approximately 2.75 nm, which is match with the consequence of TEM. Functional groups and chemical compositions of N-CDs were described by Fourier transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS). As illustrated in Fig.1f, the absorption bands at 3666 cm−1 and 3367 cm−1 are attributed to the stretching vibrations of O-H and N-H, respectively. There exist double strong peaks at 2978 cm−1 and 2899 cm−1, corresponding to the stretching vibrations of -C-H. The

Fig.2 (a) UV-Vis absorption and PL spectra of N-CDs. Inset: Images of the N-CDs aqueous solution in sunlight (left) and ultraviolet radiation (right); (b) Excitation-emission matrix for N-CDs. stretching vibration caused by the C=O functional group is presented in 1607 cm−1. Moreover, the stretching vibrations of C = N and -C-N = are located in the absorption bands at 1559 cm−1 and 1402 cm−1, respectively. In addition, the strong absorption peak at 1060 cm−1 is assigned to the C-N stretching vibration in tertiary amine-like structure. Above result demonstrates that the surface of N-CDs are rich in amino and carboxyl groups. XPS spectrum was implemented for further verification of characteristic functional groups of N-CDs. As depicted in Fig.1c, the characteristic binding energy signals of C1s, N1s and O1s exhibit three main peaks at 284.5, 400.5 and 532.1 eV, respectively. The high-resolution spectra of C1s (Fig.1d) can be decompose into three peaks at 284.6 eV (CC/C=C), 285.9 eV (C-N/C-O), 287.0 eV (COOH). As shown in Fig.1e, the spectrum of the N1s demonstrates three peaks of pyridine N, C-N-C and graphitic N-C3, which located at 398.6 eV, 399.7 eV and 400.9 eV, respectively. The O1s spectrum peaks at 531.4 eV and 532.5 eV in agreement with C-OH and C=O (Fig.S1). The results of XPS are consistent with the confirmation of FTIR. PL spectra and UV-Vis absorption were employed as powerful tools for verifying the optical characteristics of NCDs. The UV-Vis absorption in Fig.2a illustrates two intense absorption peaks at 273 nm and 448 nm, corresponding to the π−π* transition of C=C and the n–π* transition of the C=O or C−N, respectively. Moreover, according to fluorescence spectra in Fig.2a, the optimal excitation (λex) and emission (λem) wavelength were located in 478 nm and 618 nm. The images of N-CDs aqueous under 365 nm UV light irradiation is inserted in Fig.2a, and it exhibits strong orange photoluminescence. As indicated in Fig.2b, N-CDs exhibited excitation-dependent emission when the excitation wavelength changes ranging from 300 to 600 nm and the optimum emissive is located at 618 nm at the excitation wavelength of 478 nm. This property illustrated that PL properties of N-CDs mainly depended on the surface state. The QY of the as-synthesized N-CDs was calculated to be 13.9 % choosing Rhodamine B as the reference (Fig. S2). As demonstrated in Fig.S3, the fluorescence decay of N-CDs is calculated to be 3.09 ns. Comparatively, the fluorescence lifetime of CDs@Ag(I)，CDs@Ag(I)@GSH are measured to be 3.71 ns and 2.98 ns, which shows no obvious difference with N-CDs.

Fig.1 (a) TEM image, HRTEM image (top right inset) and the size distribution (top left inset) of synthesised N-CDs; (b) The AFM image of N-CDs; (c) Full XPS spectrum; (d) C1s XPS spectrum; (e) N1s XPS spectrum; (f) FT-IR.

The fluorescence fluctuations of N-CDs under different pH is described in Fig.S4. The PL intensity of N-CDs presenting irregular changes ranging from 1 to 14 and achieving higher range at pH 7 - 8, which indicated that N-CDs are suitable for biological systems. The photostability experiment of N-CDs is



Fig.3 (a) Fluorescence spectrum of N-CDs (0.1 mg·mL-1) with addition of various concentrations of Ag+. Insert: Fluorescence pictures of N-CDs in the absence and presence of Ag+ under 365 nm UV lamp illumination. (b) Relationship between the relative fluorescence intensity (I618 nm/I532 nm) of N-CDs and the concentration of Ag+ at 0-140 μM. Insert: Linearity of response. (c) UV-Vis spectrum of N-CDs (0.03 mg·mL-1) with addition of various concentrations of Ag+. (d) Relationship between the absorbance intensity of N-CDs with concentration of Ag+ at 0-50 μM. Insert: Linearity of response.

Fig.4 (a) Fluorescence spectrum of CDs@Ag(I) with addition of Fig.4 (a) Fluorescence spectrum of CDs@Ag(I) with addition of various concentrations of GSH. Insert: Photographs of CDs@Ag(I) various concentrations of GSH. Insert: Fluorescence pictures of in the absence and presence of GSH under 365 nm UV lamp CDs@Ag(I) in the absence and presence of GSH under 365 nm UV irradiation. (b) Relationship between the relative fluorescence lamp illumination. (b) Relationship between the relative intensity (I nm/I532 nm) of CDs@Ag(I) and concentration of GSH fluorescence618intensity (I nm/I532 nm) of CDs@Ag(I) and the in the range of 0-120 μM.618 Insert: Linearity of response. (c) UV-Vis concentration of GSH at 0-120 μM. Insert: Linearity of response. (c) spectrum of CDs@Ag(I) with addition of different concentration of UV-Vis spectrum of CDs@Ag(I) with addition of different GSH. (d) Relationship between the absorbance intensity of concentration of GSH. (d) Relationship between the absorbance CDs@Ag(I) with concentration of GSH from 0 to 45μM. Insert: intensity of CDs@Ag(I) with concentration of GSH at 0-45 μM. Linearity of response. Insert: Linearity of response.

illustrated in Fig. S5, which was proved by recorded the fluorescence intensity when continuously irradiated with a xenon arc lamp for 60 minutes at λex/λem= 478 nm / 618 nm. The effects of ionic strength on the fluorescence variation of N-CDs was described in Fig.S6. The spectral characteristics of N-CDs observe no significant changes at different concentrations of NaCl, which confirms the promising utilization of N-CDs in physiological salt environment.

(Fig.3b insert). Meanwhile, the limit of detection (LOD) for Ag+ was measured to be 0.45 μM (S/N=3). Moreover, the absorbance titrations also shows exceptional ExpDec1 modle: y = 0.5078*exp(-x/31.0432) + 0.4999, R2 = 0.998. A good linear response to Ag+ was determined in the range of 10 - 40 μM with a detection limit (3σ) of 0.27 μM, the linear regression equation are (A0-A)/A0=0.0077C+0.0722, R2=0.9844, where A0 and A are the absorbance intensities of N-CDs in the absence and presence of Ag+ (Fig.3c and Fig.3d). Above results displayed that the N-CDs can be probably applied as a selective nanosensors for Ag+ via dual-signal sensing model.

Ratiometric Detection of Ag+. As shown in Fig.4, the ratio of fluorescence intensity (I618 nm/I532 nm) of N-CDs interacts with metal ions at a concentration of 10 mM (K+, Ca2+, Na+, Mg2+, Al3+, Zn2+, Co2+, Cr6+, Fe3+,Cu2+, Hg2+, Cr3+, Mn2+, Pb2+, Ni2+, Ba2+, Fe2+, Cd2+, Sn2+, Ag+) were collected and recorded. As demonstrated in Fig.S7, the ratio of I618 nm/I532 nm is significantly reduced with the introduction of Ag+, while the other metal ions show negligible effect. The result reveals that the high specificity of N-CDs toward Ag+ over competitive metal ions, which indicating that the proposed ratiometric nanosensor may be further utilized in fluorescent based biosensing for sensitive detection of Ag+. Upping introduce of Ag+ into N-CDs system, the PL intensity at 618 nm declines regularly accompanying with a ratio enhancement at 532 nm. Interestingly, the fluorescence of N-CDs solution changes from orange to green (Fig.3a). As shown in Fig.3b, the ratio of fluorescence intensity at 618 nm and 532 nm were well fitted using ExpDec1 modle with changing of Ag+ concentration in the area of 0 - 140 μM, the fitting equation is y=2.7783*exp(-x/43.0657) + 0.8542, R2 = 0.998. In addition, the value of I618 nm/I532 nm shows a linear relationship with the Ag+ concentration in the range of 30 - 90 μM, the linear regression equation is I618 nm/I532 nm = 0.0161C + 2.5876, R2 = 0.9723, where C is the concentration of Ag+

Ratiometric Detection of GSH. Various amino acids at a concentration of 10 mM (Phe, Lys, Thr, Hcy, Cys, Asp, Tyr, Ser, Ile, Glu, Gln, Leu, His, Gly, Trp, Val, Pro, Arg, Ala, Met, GSH) was introduced into the N-CDs solution (0.10 mg∙mL-1) in the presence of 140 μM Ag+ respectively. As shown in Fig.S8, CDs@Ag(I) system demonstrates specific recognition to GSH over other competitive substance. Moreover, the influence of related active sulfur/oxygen on the relative fluorescence intensity of CDs@Ag(I) were explored in Fig.S9, which demonstrated negligible variation on CDs@Ag(I) system. The producing N-CDs can be utilized as dualfunctionalized sensing platform for specificity detection of GSH. The different concentrations of GSH was added into the CDs@Ag(I) (0.1 mg·mL-1 N-CDs with 140 μM Ag+) solution in the range of 0-120 μM (Fig.4a). Ag+ was escaped from the surface of N-CDs, the fluorescence intensity at 618 nm recovers gradually with increasing amounts of GSH, while the fluorescent at 532 nm reduce to almost the same fluorescence intensity as before. Meanwhile, the value of I618 nm/I532 nm exhibits a good linear correlation (I618 nm/I532 nm = 0.0232C + 0.75, R2 = 0.999) with varying of GSH in the range of 10-100 μM, and the limit of detection is calculated to be 0.38 μM


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Fig.5 Calculated CIE coordinates from the PL spectra of CDs, CDs@Ag(I) and CDs@Ag(I)GSH. (Fig.4b). Similarly, an excellent linear correlation to GSH at concentration of 0.36 - 30 μM was obtained via UV-Vis spectrophotometer, the linear regression equation is (A0A)/A0=0.0198C + 0.0447, R2 = 0.9749, where A0 and A are the absorbance intensities of CDs@Ag(I) in the absence and presence of GSH. The limit of detection was determined as 0.12 μM (Fig.4c and Fig.4d). The fluorescence emission of CDs, CDs@Ag(I) and CDs@Ag(I)@GSH are further confirmed with CIE coordinates (0.5948, 0.4046), (0.4401, 0.5452) and (0.5838, 0.4154) and the results is described in Fig.5, which further demonstrates the immense potential of implemented NCDs as a fluorescence visualization biosensing platform in biological systems. To further understand the interaction mechanism, FTIR spectroscopy, DLS, and zeta potential were operated to characterize the change of N-CDs during the response of Ag+ and GSH. Contradistinction with the FTIR spectrum of N-CDs (Fig.S10), the sharp decrease in the peaks at 1607 and 1559 cm−1 of CDs@Ag(I) confirms that probable binding of Ag+ to the C=O or C=N functional groups of the N-CDs. As shown in Fig.S11, the hydrodynamic diameter of N-CDs, CDs@Ag(I) and CDs@Ag(I)GSH are 78.82, 24.35and 68.06 nm, respectively. When Ag+ are added into the N-CDs system, the thickness of the electrical double layer of N-CDs is destroyed, resulting in a smaller hydrodynamic diameter of CDs@Ag(I). Introducing the GSH into CDs@Ag(I) system, Ag+ was removed from the surface of N-CDs and the hydrodynamic diameter returned to 68.06 nm. The zeta potential also provided the consistent results (Fig.S12). The zeta potential of N-CDs aqueous solution increased dramatically from -17.5 mV to + 1.28 mV in the presence of Ag+, probably owing to the consumption of the carboxyl or other negative groups on the surface of N-CDs with the introducing of Ag+ and it further confirms that the successful combination of Ag+ with N-CDs. Combined with UV-Vis spectra where a new weak absorption peak was emerged at 335nm in the CDs@Ag(I) system, triggering by N - Ag ligand-to-metal charge transfer (Insert in Fig.3c). All this results demonstrating there may form a stable

Fig.6 The confocal laser scanning microscope of Ag+ and GSH in living SMMC7721 cells. (a) SMMC7721 cells pretreated with NEM (1.0 mM) for 30 min and then incubated with N-CDs (0.3 mg·mL-1) for 30 min. (b) SMMC7721 cells in the presence of CDs pretreated with 25 μL of Ag+ (0.01 M) for 2h. (c) 25 μL of GSH (0.01 M) was introduced into above system. (d, e) Cys and Hcy were introduced into CDs@Ag(I) pretreated SMMC7721cells. Channel 1: λem = 500-560 nm, Channel 2: λem = 560-650 nm, from left to right: Channel 1 (green channel), Channel 2 (orange channel), Overlay, Ratio.

complex between N-CDs and Ag+ and it further results in a rising fluorescence peak emerges at 532 nm accompanying with the intensity at 618 nm declines accordingly in a ratiometric way. Moreover, Ag+ has a high affinity preference to biothiol as a thiophilic metal ion. According to the literature, we conclude that the influence in the size of GSH and the nucleophilic ability of the GSH to CDs@Ag(I) may lead to the specific discrimination of GSH from Hcy and Cys48. After introducing GSH into the above CDs@Ag(I) system, Ag+ can be competitively captured by GSH in consequence of its stronger binding ability with GSH and the fluorescence intensity of CDs@Ag(I) at 618 nm is significantly restored along with the fluorescence decreased at 532 nm, which realizing the ratiometric fluorescence response to GSH. Therefore, the NCDs can implemented as a powerful tool for fluorescence visualization sensing of Ag+ and GSH accompanied by reversible changes with orange and green fluorescence. Biocompatibility study. Human Hepatic carcinoma SMMC-7721 cells was selected as a standard to estimate the cytotoxicity of N-CDs (Fig.S13). It indicates that nearly 88% cells survived after culturing SMMC-7721 cells with different concentrations of N-CDs (0 - 500 μg∙mL-1). In addition, the cytotoxicity of CDs@Ag(I) on SMMC7721 cells viability was tested. As shown in Fig.S14, incubating N-CDs with different concentration of Ag+ ranging from 0 to 100 μM, the cell



viability remains greater than 80%. All this results demonstrating that the prepared nanosensors possess ultra-low toxicity and can be further applied in biological systems. Cellular imaging. The bioimaging applications of N-CDs was evaluated by SMMC7721 cells in vitro imaging. As exhibited in Fig.S15 (a-c), the N-CDs can be rapidly uptake by SMMC7721 cells and displays blue, green and orange multicolor emission properties under excitation of 405, 488 and 561 laser, respectively. The merged images of three panels is presented in Fig.S15 (d-e), indicating N-CDs hold great potential for bioimaging applications. Pretreatment of SMMC7721 cells with a typical thiol blocker N-ethylmaleimide (NEM, 1.0 mM) for 30 minutes before incubation with N-CDs for internal depletion of intracellular thiol. The ratio fluorescence imaging of SMMC7721 cells incubated with N-CDs were achieved by collecting dual emission channels: (1) green (λem = 500-560 nm) and (2) orange (λem = 560-650 nm) channels (Fig.6) under excitation at 488 laser. As demonstrated in Fig.6a, there initially exist weak green fluorescence in channel 1 accompanying with bright orange fluorescence in channel 2. With addition of Ag+, the fluorescence intensity in green channel gradually increased, while the fluorescence intensity of orange channel decreased evidently (Fig.6b). Conversely, the green fluorescence in channel 1 was diminished concomitant with a sharp increase in the orange channel with the addition of GSH (Fig.6c). The fluorescence ratio image (I Channel1/I Channel2) was obtained using Image-J software, inferring the potential application of N-CDs in cellular imaging. In addition, the overlay of channel 1 and channel 2 exposed a distinct color variation with the sequential addition of Ag+ and GSH. However, other interfering substances such as Cys (Fig.6d) and Hcy (Fig.6e) did not contribute to the fluorescence recovery of CDs@Ag(I), which confirm the superiority of N-CDs for specifically recognizing Ag+ and GSH in biological system. CONCLUSIONS In summary, ratiometric fluorescent N-CDs with longwavelength emission were fabricated through a facile hydrothermal method. The obtained N-CDs were demonstrated as a biosensing platforms for sequential detection Ag+ and GSH based on ratiometric fluorescence changes at 618 nm and 532 nm along with reversible fluorescence variation of orange to green. It hold excellent advantage in enhancing the sensitivity of trace levels of analyst and built-in correction of environmental impact, which can implemented as a promising biosensors for intracellular determination of Ag+ and GSH. In addition, the N-CDs possesses excellent dispersion, favorable biocompatibility and low toxicity, which can achieve ratiobased fluorescence bioimaging of N-CDs in SMMC-7721cells based on the change in ratio fluorescence intensity (I618 nm/I532 nm). The N-CDs exhibit exceptional fluorescence reversibility and photo stability against potentially interfering substances. It is anticipated that the N-CDs can regard as a favorable biosensors to construct a dual-sensing platforms for tracking of Ag+ and GSH in clinical diagnosis and other biomedical fields.

Optimize the optical properties of ratiometric fluorescence N-CDs, the exhaustive structural characterization, validation of specificity of N-CDs to Ag+ and GSH and complementary graphical illustrations for application of N-CDs in biological systems. AUTHOR INFORMATION Corresponding Author *Chuan Dong; Email address: [email protected]; Tel: +86-3517018613; Fax: +86-351-7018613 Present Addresses Author Contributions The manuscript was written through contributions of all authors. /All authors have given approval to the final version of the manuscript. Funding Sources This work was financially supported by the National Natural Science Foundation of China (No. 21874087, 21475080 and 21575084), Shanxi Province Hundred Talents Project and Shanxi Province Graduate Education Innovation Project (2017BY006). ACKNOWLEDGMENT We appreciate Scientific Instrument Center of Shanxi University for us to identify samples. REFERENCES

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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

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Gao, X.; Lu, Y.; Zhang, R.; He, S.; Ju, J.; Liu, M.; Li, L.; Chen, W., One-pot Synthesis of Carbon Nanodots for Fluorescence Turn-on Detection of Ag+ Based on the Ag+-induced Enhancement of Fluorescence. Journal of Materials Chemistry C 2015, 3, 2302-2309. Lin, Y. H.; Tseng, W. L., Highly Sensitive and Selective Detection of Silver Ions and Silver Nanoparticles in Aqueous Solution Using an Oligonucleotide-based Fluorogenic Probe. Chemical Communications 2009, 43, 6619-6621. Cayuela, A.; Soriano, M. L.; Kennedy, S. R.; Steed, J. W.; Valcárcel, M., Fluorescent Carbon Quantum Dot Hydrogels for Direct Determination of Silver Ions. Talanta 2016, 151, 100-105. Liu, G.; Xuan, C.; Feng, D. Q.; Hua, D.; Liu, T.; Qi, G.; Wang, W.; Liu, G.; Xuan, C.; Feng, D. Q., Dual-modal Fluorescence and Light-scattering Sensor Based on Water-soluble Carbon Dots for Silver Ions Detection. Analytical Methods 2017, 9, 5611-5617. Sung, Y. M.; Wu, S. P., Highly Selective and Sensitive Colorimetric Detection of Ag(I) using N-1-(2mercaptoethyl) Adenine Functionalized Gold Nanoparticles. Sensors & Actuators B Chemical 2014, 197, 172-176. Bian, S.; Shen, C.; Qian, Y.; Liu, J.; Xi, F.; Dong, X., Facile Synthesis of Sulfur-doped Graphene Quantum Dots as Fluorescent Sensing Probes for Ag+ ions Detection. Sensors & Actuators B Chemical 2017, 242, 231-237. Alan, C.; Aleksandar, R.; Shane, P.; Eric, B.; Ernŏ, P., Rational Design of Potentiometric Trace Level Ion


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TOC 254x190mm (96 x 96 DPI)


One-step synthesis of lable-free ratiometric fluorescence carbon dots

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