Preparation of an Efficient Ratiometric Fluorescent Nanoprobe (m-CDs

Nov 2, 2017 - †Institute of Nanomedicine and Nanobiosensing, MOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial...
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Preparation of an Efficient Ratiometric Fluorescent Nanoprobe (m-CDs@[Ru(bpy)3]2+) for Visual and Specific Detection of Hypochlorite on Site and in Living Cells Yuanjin Zhan, Fang Luo, Longhua Guo, Bin Qiu, Yuhong Lin, Juan Li, Guonan Chen, and Zhenyu Lin ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00601 • Publication Date (Web): 02 Nov 2017 Downloaded from http://pubs.acs.org on November 3, 2017

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Preparation of an Efficient Ratiometric Fluorescent Nanoprobe (mCDs@[Ru(bpy)3]2+) for Visual and Specific Detection of Hypochlorite on Site and in Living Cells Yuanjin Zhan,a, 1 Fang Luo,b, 1 Longhua Guo,a* Bin Qiu,a* Yuhong Lin,a Juan Li,a Guonan Chen, and Zhenyu Lina a

Institute of Nanomedicine and Nanobiosensing; MOE Key Laboratory for Analytical Science of Food Safety and Biology; Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety; College of Chemistry, Fuzhou University, Fuzhou, 350116, China b College of Biological Science and Engineering, Fuzhou University, Fuzhou, Fujian 350116, China ABSTRACT: Hypochlorite (ClO-) is one of the most important reactive oxygen species (ROS), which plays an important role in sustaining human innate immunity during microbial invasion. Moreover, ClO- is a powerful oxidizer for water treatment. The safety of drinking water is closely related to its content. Herein, m-phenylenediamine (mPD) is used as a precursor to prepare carbon dots (named m-CDs) with highly fluorescent quantum yield (31.58% in water), and our investigation shows that the strong fluorescent emission of m-CDs can be effectively quenched by ClO-. Based on these findings, we developed a novel fluorescent nanoprobe (mCDs) for highly selective detection of ClO-. The linear range was from 0.05 to 7 µM (R2 = 0.998), and the limit of detection (S/N = 3) was as low as 0.012 µM. Moreover, a portable agarose hydrogel solid matrix-based ratiometric fluorescent nanoprobe (mCDs@[Ru(bpy)3]2+) sensor was subsequently developed for visual on-site detection of ClO- with the naked eyes under a UV lamp, suggesting its potential in practical application with low cost and excellent performance in water quality monitoring. Additionally, intracellular detection of exogenous ClO- was demonstrated via ratiometric imaging microscopy.

Keywords: hypochlorite; carbon dots (CDs); ratiometric fluorescent nanoprobe; m-CDs@[Ru(bpy)3]2+; agarose hydrogel solid matrix; ratiometric imaging microscopy Hypochlorite (ClO-) is a prominent member of reactive oxygen species (ROS)1-3 and their intimate effects on health of humans and animals, play important roles in toxicology and pathology. Endogenous ClO- is generated by the reaction between hydrogen peroxide (H2O2) and chloride ions (Cl-) via catalyzer (heme enzyme myeloperoxidase (MPO)).4, 5 Abnormal levels of endogenous ClO- are associated with certain diseases such as cardiovascular diseases, neuron degeneration, lung injury, atherosclerosis, arthritis, and cancer.6-8 Meanwhile, ClO-, a powerful oxidizer, has been widely used in water disinfection, deodorization, blanching, and other numerous manufacturing processes. In water treatment, the concentration of ClO- must be strictly controlled. Because a very small amount of ClO- cannot kill pathogenic bacteria and hence causes many hazards. Meanwhile, a large amount of ClO- may produce many undesirable byproducts, especially trihalomethane (THMs),9, 10 which is harmful to human beings and animals and can result in diseases such as reproductive failure,11 renal disease, atherosclerosis,12 and cancers.13 Therefore, it is necessary to monitor and control ClO- level in drinking water and vivo. To date, many sensors based on fluorophores have been developed to detect ClO-, including luciferin,14 fluorescein,15 rhodamine,16, 17 NIR Pdots,18 boron-dipyrromethene (BODIPY),19 lanthanide complexes,20 and other organic molecular chemodosimeters.21-24 In addition, Tian et al. have utilized surface-enhanced Raman scattering (SERS) nanoprobe

for imaging and real-time detection of ClO- and GSH in live cells upon oxidative stress.25 Additionally, continuing efforts have been focused on building an excellent probe for the detection of ClO-. Among them, the fluorescence method is an attractive alternative in terms of sensitivity, spatial and temporal resolution, and simplicity of use in living cells and organisms. However, single-emission fluorescence probe usually suffers from many adverse effects originating from instrumental efficiency, environmental conditions, and the concentration of probe molecules. To eliminate aforementioned adverse factors and obtain accurate results, ratiometric fluorescence is precise in response to the trend of the times. Ratiometric fluorescence technique for constructing chemo/bio sensors have attracted increasing attention owing to the improved sensitivity at trace quantity levels of analyte and built-in correction for environmental effects, as well as improved semi-quantitative visualization capability by displaying continuous color changes.26-28 Novel nanomaterials have attracted more attention in biological analysis and detection. Carbon dots (CDs) have emerged as a kind of novel photoluminescence (PL) nanomaterial with low cost,29 low toxicity, high photoluminescence,30 and good biocompatibility.31 Owing to these outstanding merits, CDs have been widely used for detecting metal ion, enzymatic activity, imaging and other small molecules.32 Typically, CDs of less than 10 nm has an sp2 or amorphous carbon framework, and the fluorescence properties of CDs is attribut-

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ed to structural defects induced by finite size and element doping (e.g., O, N).33-35 Herein, we developed a facile one-step hydrothermal approach to prepare highly fluorescent carbon dots (named mCDs) with m-phenylenediamine (mPD) as a precursor, and we found its PL quantum yields (PLQY) were as high as 31.58 % when tested in water. In this case, the prepared m-CDs were explored as a fluorescent probe for highly selective analysis of ClO-. We proposed a novel ratiometric probe for the visual observation of fluorescence color changes induced by ClOwithout the need of elaborate equipment. As illustrated in Scheme 1, we use m-CDs and Ru(bpy)32+ to design the ratiometric probe because they not only afford the singleexcitation/multiple emissions but also provide high sensitivity and selectivity. Under optimal conditions, in the presence of different amounts of ClO-, the probe displays continuous color changes from cyan to red due to the variations of the dual emission intensity ratios, which can be clearly observed with the naked eye. These ratio and color change can be used for both qualitative recognition and quantitative analysis of ClO-. Moreover, a portable agarose hydrogel solid matrix-based sensor has been developed by mixing and solidifying mCDs@[Ru(bpy)3]2+ probe solution into agarose hydrogel using a microwave oven. Visual fluorescence detection of ClO- with the naked eye was achieved under a UV lamp, suggesting its potential in practical application for the on-site detection of ClO-. Compared with a single wavelength emissive fluorescent probe, the ratiometeric method exhibits significantly enhanced visual detection sensitivity. Additionally, intracellular detection of exogenous ClO- has been demonstrated via ratiometric imaging microscopy.

Scheme 1. Illustration of the ratiometric fluorescent nanoprobe for visual detection of ClO-.

MATERIALS AND METHODS Materials. m-Phenylenediamine (mPD) was purchased from Sinopharm Group Co., Ltd. (Shanghai, China). NaClO•5H2O was purchased from Wako Pure Chemical Industries, Ltd. (Japan). Ru(bpy)3Cl2•6H2O was purchased from Energy Chemicals Reagent Co., Ltd. (Shanghai, China). Agarose was purchased from GEN-VIEW SCIENTIFIC INC. All reagents were of analytical grade, and used as received. Ultrapure water was obtained from a Millipore purification system (18.2 MΩ•cm, Millipore, USA) and used for the project. Instruments. Scanning electron microscope (SEM), transmission electron micrograph (TEM), and atomic force micro-

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scope (AFM) images were obtained by using SU8010 SEM system (HITACHI, Japan), A JEM-2100 TEM instrument (JEOL, Japan), and Agilent 5500 AFM/SPM system (Agilent, USA), respectively. 1H-NMR was surveyed by using AVANCE III HD 400 MHz (Bruker, Switzerland). X-ray photoelectron spectroscopic (XPS) was performed using an ESCALAB 250 spectrometer (Thermo-VG Scientific, USA) with an ultrahigh vacuum generator. Fourier transform infrared spectroscopy (FTIR) spectra were recorded using a NICOLET-6700 spectrometer (Thermo scientific, USA). UVvis absorption spectra were obtained with a UV-2310 UV-visNIR spectrophotometer (Tianmei, China). Fluorescence lifetime was measured with a F900 fluorescence spectrometer (Edinburgh Instruments Ltd., U.K.). Fluorescence spectra were recorded on a F-4600 fluorescence spectrophotometer (HITACHI, Japan) equipped with a Xenon lamp. The fluorescence images of stained cells were acquired with a fluorescence confocal microscope (Nikon, C2/C2si, λex = 488 nm, λex = 543 nm). Synthesis of m-CDs and m-CDs@[Ru(bpy)3]2+. mPhenylenediamine (mPD, 0.90 g) was firstly dissolved in ethanol (90 mL), and then the solution was transferred into poly(tetrafluoroethylene)-lined autoclaves. After being heated in oven (180 ºC, 12 h) and cooled down to room temperature naturally, the crude products were then purified with a silica column chromatography using mixtures of methylene chloride and methanol as eluents. After being removed solvents and further dried under vacuum, m-CDs could be finally obtained.36 Briefly, m-CDs@[Ru(bpy)3]2+ fluorescent nanoprobe was synthesized by mixing 40 µg•mL-1 of Ru(bpy)3Cl2·6H2O (red emission) with 20 µg•mL-1 of m-CDs (cyan emission) in a volume ratio of 1:6. Preparation of Portable m-CDs@[Ru(bpy)3]2+ Agarose Hydrogel Solid Matrix. A portable agarose hydrogel solid matrix for ClO- was prepared as follows: agarose (0.2 g) was completely dissolved in boiling m-CDs@[Ru(bpy)3]2+ solutions (20 mL) with stirring. After uniform blending, the mixture solution (200 µL) was transferred into the cap of the microcentrifuge tube. The hydrogel was shaped after drying under ambient temperature for 3 min, and the hydrogel solid matrix was stored in the refrigerator at 4 ℃ before use. The hydrogel were stable for at least two weeks. Fluorescence Assay of ClO-. Fluorescence studies were carried out in a 1 cm ×1 cm quartz cell, where m-CDs (50 µL, 20 µg•mL-1), phosphate buffered solution (PBS, 50 µL, 0.1 M, pH = 7.0), and different concentrations of ClO- solution (100 µL) were added sequentially and mixed for fluorescence measurements (λex = 384 nm, λem = 488nm). Analysis of Real Sample. A local tap water sample was collected (Fuzhou, China) and analyzed for ClO- through both the N,N-diethyl-p-phenylenediamine (DPD) colorimetric procedure and the proposed method without dilution. To ensure the accuracy of the results, the water samples were analyzed as soon as collected in consideration of their poor stability. Cell Culture and Imaging. For detection of exogenous HOCl, the cultured HeLa cells in a T-75 flask were washed three times with Krebs-Ringer phosphate buffer (KRP buffer, 114 mM NaCl, 4.6 mM KCl, 2.4 mM MgSO4, 1.0 mM CaCl2, 15 mM Na2HPO4/NaH2PO4, pH = 7.4) and then incubated with the KRP buffer containing m-CDs (20 µg•mL-1) and m-

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CDs@[Ru(bpy)3]2+ for 1 h at 37 °C in a 5% CO2/95% air incubator, respectively. After being washed three times with KRP buffer, the cells were cultured for another 15 min with the KRP buffer containing 10 µM of NaOCl. The cells were washed five times carefully with KRP buffer and then subjected to the luminescence imaging measurement. RESULTS AND DISCUSSION Characterization of m-CDs. m-CDs solution exhibits a long-term homogeneous phase without any noticeable precipitation at room temperature. Figure 1A shows the TEM image of the monodispersed m-CDs and the size distribution of m-CDs (about 5-6 nm). The corresponding AFM image (Figure 1B) shows that all of the m-CDs are monodispersed and exhibited similar particle heights of approximately 1.5 nm (Figure 1C). FT-IR spectra were employed to characterize the functional groups and chemical bonds on m-CDs. As shown in Figure S-1A, in comparison to the initial materials mPD, some new characteristic peaks emerged at about 2805–2958, 1385.9, and 1245.8 cm-1 for mCDs, respectively. This outcome implies that decomposition, intermolecular cyclization, and polymerization reactions occur during the formation of m-CDs.36 1H NMR was carried out to speculate the position and the types of the hydrogen atom in the carbon skeleton. As shown in Figure S-1B, three different chemical shifts of m-CDs were detected by 1H NMR spectra (400 MHz, in D2O), which reveals that m-CDs possesses 12 hydrogen atoms and further verifies above-mentioned speculation. Although these reaction processes were suggested in the formation of similar CDs prepared from oPD,37, 38 the actual mechanism remains unclear. m-CDs aqueous solution shows bright cyan emission under excitation of 365 nm UV light. The emission wavelength of mCDs was excitation independent. The maximum excitation wavelength and maximum emission wavelength of m-CDs were 384 and 488 nm, respectively (Figure 1D). m-CDs exhibited an apparent UV-vis absorption band centering around 384 nm, which corresponds well to its excitation spectra. The PLQY (see supporting information) and PL lifetimes of mCDs were measured to be 31.58 % and 8.80 ns, respectively. Interestingly, we observed different fluorescence properties of m-CDs in ethanol and water with solvent effect and red-shift effect. Comparing to m-CDs dissolved in ethanol, m-CDs with higher PLQY, longer PL lifetimes and larger stokes shifts in water (Figure S-2).

Figure 1. (A) TEM of m-CDs. The inset image shows the size distribution of m-CDs in the TEM image. (B) AFM images of m-CDs. (C) The height of m-CDs. (D) UV–vis absorption (Abs, green), Ex (red), and Em (black) spectra. Inset of (D) shows the corresponding photographs of the m-CDs dispersed in water taken under visible (left) and 365 nm UV (right) lights, respectively.

Establishment of m-CDs Based Fluorescent Sensor for ClO-. Such a highly fluorescent m-CDs motivated us to explore its promising application in fluorescent sensing. Inspiringly, we found that its strong fluorescence was dramatically suppressed after contacting with ClO- (Figure 2A), thus further efforts were made toward the establishment of m-CDs-based ClOsensor. To understand the response rate of the fluorescence (FL) signal of m-CDs to the ClO-, the time-dependent FL changes upon addition of 10 µM ClO- was first monitored. As shown in Figure 2B, the fluorescence intensity of m-CDs at 488 nm rapidly reduced about 90% within 5 min compared with the original value when 10 µM of ClO- was incorporated. To ensure the consistency of experimental conditions, 5 min was selected as the collection time of fluorescence data.

Figure 2. (A) UV and FL spectra of 20 µg•mL-1 of m-CDs solution in the absence and presence of 0 (black line), 5 (red line), and 10 (gree line) µM ClO-, respectively. (B) Time-dependent FL response of the m-CDs (20 µg•mL-1) to ClO- (10 µM) in PBS (pH = 7.0).

Another key factor for the sensing system is the pH value of solution. On one hand, the pH value may affect the FL activity of m-CDs. As shown in Figure 3 (red columns), in alkaline solutions, FL intensity of m-CDs was relatively low and obviously pH dependent, which is unfavorable for developing a sensitive and stable sensing method. In contrast, the FL intensity of m-CDs reaches the maximum value and keeps relatively stable when pH is lower than 7.0, indicating that neutral and acidic conditions might be suitable for this sensing system. On the other hand, pH value also may affect the forms of ClO- in water.39 ClO- exists mainly as hypochloric acid (HClO) in neutral and weakly alkaline solutions (pH 3-9) and hypochlorite anion (ClO-) in strong alkaline solutions (pH > 9). As shown in Figure 3 (blue and green columns), the quenching effect of ClO- (10 µM) on the FL intensity of m-CDs decreased with the increasing of pH value, and quenching efficiency F0/F of ClO- on the FL intensity reaches maximum value at pH = 7.0. Considering the possible application in living cells, pH = 7.0 was chosen as the optimized pH throughout subsequent studies.

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cies existing in natural water samples and is suitable for the selective analysis of ClO- in real samples.

Figure 3. FL response of m-CDs (20 µg•mL-1) in the absence and presence of ClO- (10 µM) at different pH values. Error bars indicate standard deviations of three independent measurements.

Under the optimized conditions, the linear range of the sensing system was investigated. As shown in Figure 4, with the increasing of ClO- concentration, the fluorescence intensity of m-CDs at 488 nm decreased gradually. There was a good linear relationship between the quenched fluorescent intensity of m-CDs and the concentration of ClO- in the range of 0.05-7 µM (R2 = 0.998), and the limit of detection (LOD) was as low as 0.012 µM (S/N = 3). This outcome demonstrates that the proposed method is much better than many other sensors (Table S-1).

Figure 4. (A) The FL response of m-CDs (20 µg•mL-1) upon addition of various concentrations of ClO- in PBS (pH = 7.0). From top to bottom (a-p): 0, 0.05, 0.1, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 15 µM, respectively. (B) Stern-Volmer plot of FL quenching of the m-CDs by ClO-. The inset shows Standard curve (equation: FL = 499.5-54.80 CClO-; R2 = 0.998) for the determination of ClO- concentration. Error bars indicate standard deviations of five independent measurements.

Before being applied in real sample detection, selectivity is also an important evaluation parameter. Effects of some common ions (F-, Cl-, Br-, I-, SO42-, CO32-, NO3-, PO43-, Na+, K+, Ca2+, Mg2+, NH4+, Zn2+, Mn2+, Fe3+, Cu2+, Al3+, Ba2+, Co2+, and Ni2+) and some heavy metal ions (Pb2+, Hg2+, Cd2+, Cr3+, and Ag+) were also investigated. As shown in Figure 5A, these ions had nearly no interference even at a high concentration (common ions were 1 mM; heavy metal ions were 100 µM). Apparently, the present sensing system exhibits excellent selectivity for the detection of ClO-. No obvious fluorescence intensity changes were observed for m-CDs in the presence of other interferences including ROS, RNS, glucose, AA, biothiols, metal ions, and amino acids (Figure 5B). Apparently, the results confirm that the presented sensing system exhibits high selectivity for ClO- against the most potential interfering spe-

Figure 5. (A) Selectivity of the m-CDs-based sensor for ClOover other ions in PBS (pH = 7.0): concentrations of m-CDs and ClO- were 20 µg•mL-1 and 10 µM, respectively; concentrations of other common ions were all 1 mM; concentrations of heavy metal ions were 100 µM (from a to z: blank, ClO-, Ag+, Al3+, Ba2+, Cd2+, Co2+, Cr3+, Cu2+, Fe3+, Hg2+, Mn2+, Ni2+, NH4+, Pb2+, Zn2+, CO32-, NO3-, PO43-, SO42-, CaCl2, MgCl2, KI, NaF, NaCl, NaBr). (B) Fluorescence responses of m-CDs (20 µg•mL-1) toward ClO- (10 µM) and other various interferences (from a to z) under PBS (pH = 7.0): blank, ClO-, H2O2, •OH, O2-, 1O2, NO2-, NO•, ONOO-, SO32-, NO3-, IO3-, K+, Na+, Ca2+, Mg2+, Zn2+, GSH, glucose, ascorbic acid (AA), glutamate (Glu), alanine (Ala), valine (Val), threonine (Thr), lysine (Lys), and leucine (Leu). Error bars indicate standard deviations of three independent measurements.

On the basis of the outstanding characteristics of the proposed method, the applicability of this proposed method for detection of ClO- in water was examined. We directly employed m-CDs to measure the concentration of ClO- in a local tap water sample from our laboratory faucets. The results show that the concentration of free residual ClO- in the tap water sample was 6.21 ± 0.05 µM (n = 10, confidence level of 95 %). The value is consistent with the result obtained by the DPD colorimetric method40-42, which was 6.32 ± 0.11 µM (n = 10, confidence level of 95 %). All the above results strongly revealed that the proposed method is capable to detect ClO- in real samples with high reliability and accuracy. Possible Mechanism of the Sensing System. It is well-known that ClO- is a powerful oxidizer and fluorescent probes can serve as reducer of ClO- in most of the ClOsensing systems. Hence, these redox-based ClO- sensing systems seriously suffered from the interferences of those oxidizers which have a stronger oxidation capability than ClO-, such as H2O2, MnO4-, Cr2O72-, ClO4-, and S2O82-. In this regard, mCDs-based sensor showed complete different result for the interacting with ClO-. As presented in Figure 6A, it is clear that the fluorescence intensity of m-CDs substantially quenched ~90% in case of 10 µM of ClO- was incorporated. Whereas other oxidizer (five-fold concentration of ClO-) could not induce marked inhibition of the fluorescence intensity (except for the KMnO4, as shown in Figure S-3, there were large-areas overlap between UV-vis spectra of KMnO4 and photoluminescence emission spectrum of m-CDs). One reasonable explanation for these findings is that it is not oxidation mechanism plays the key role in this sensing system. It is not clear what kind of interaction existing between mCDs and ClO-, but we can ensure the recovery results from the

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decomposition of ClO- and it could be further verify through the following study on the influence of illumination. The reaction mixture A−D (Figure 6B) all contained 5 µM ClO-. The fluorescence intensity of reaction mixture A was collected immediately after ClO- incorporation. Before measuring, reaction mixture B was placed in darkness for 2 h and reaction mixture C was exposed under 365 nm UV lamp for 2 h, respectively. As for reaction mixture D, the same amount of ClO- was exposed under 365 nm UV lamp with an illumination time of 2 h then added into m-CDs and recorded the photoluminescence spectrum immediately. Blank was collected upon addition of deionized water and was exposed under 365 nm UV lamp for 2 h. All corrected F/F0 values were based on the value of Blank. As shown in Figure 6B the value of reaction mixture B was close to reaction mixture A while the value of reaction mixture C was close to reaction mixture D. This result strongly indicated that ClO- exists in a stable state without light and favorably induces fluorescence quenching, but once exposed to light, the decomposition of ClO- leads to the fluorescence recovery.

Figure 6. (A) Fluorescence emission spectra of m-CDs upon addition of A) deionized water, B)10 µM NaClO, C) 50 µM H2O2, D) 25 µM KMnO4, E) 50 µM KMnO4, F) 50 µM K2Cr2O7, G) 50 µM NaClO4 and H) 50 µM (NH4)2S2O8. (B) The fluorescence quenching at various stages with the addition of 5 µM ClO-. Error bars indicate standard deviations of three independent measurements.

Moreover, XPS spectra of m-CDs before and after the addition of ClO- (Figure S-4) indicated that organochlorine bond formed between m-CDs and ClO-. Meanwhile, UV-vis and FL response of m-CDs upon the addition of different concentrations of ClO- were also studied (Figure 2A), from which observed that there was slight blue shift of the absorption peak assigned to the n-π* transition of the C-N/C=N band at about 384 nm.43 These results suggest that weak interaction exists between m-CDs and ClO-, but it is too weak to destroy the crystalline structure of m-CDs. To further explain the fluorescence quenching phenomenon, time-resolved fluorescence decay assays in the absence and presence of ClO- were performed. The results were presented in Figure S-5A, in which the fluorescence lifetime of m-CDs was shortened from 8.80 to 8.18 ns after the addition of ClO(5 µM), this outcome implies that the fluorescence quenching of m-CDs triggered by ClO- could be mainly due to the dynamic quenching effect, rather than static quenching effect. Consequently, a possible mechanism of energy transfer for the fluorescence quenching of m-CDs induced by ClO- was proposed. It is well-known that the amino group is an important functional group and used as a active binding site to bind with other ions44, 45; therefore, it is extremely necessary to test whether the amine group plays the crucial role in the sensing

system. Besides m-CDs, the quenching effect of ClO- on others C-dots (include o-CDs36, p-CDs36, N,S-dots46, graphene quantum dots47, and MCBF-CQDs48) were also examined. Interestingly, comparing to C-dots (N,S-dots, graphene quantum dots, and MCBF-CQDs) without amine group, C-dots (mCDs, o-CDs and p-CDs) with amine group all have response to ClO- in varying degrees, particularly for m-CDs (Figure S5B). Eloquently demonstrating that the amine group is the key binding site of m-CDs in responding to ClO-. To sum up, these results suggest that the sensing system is not oxidation mechanism and the quenching process is dynamic quenching effect. The amine group is the key binding site of m-CDs in responding to ClO- and formed organochlorine bond. Scheme S-1 of the supporting information shows the schematic of the whole process for ClO- sensing. It is inferred that amine groups to build N-H···O-Cl hydrogen bonding, which construct a bridge for energy migration from m-CDs to ClO-, ultimately triggering the fluorescence quenching phenomenon. The fabrication of ratiometric fluorescent nanoprobes (m-CDs@[Ru(bpy)3]2+) for Visual Detection of ClO-. To achieve semi-quantitative visualization capability, we further developed a fluorescence ratiometric method for the detection of ClO-. Briefly, 40 µg•mL-1 of Ru(bpy)3Cl2·6H2O (red emission) was mixed with 20 µg•mL-1 of m-CDs (cyan emission) in a volume ratio of 1:6. The FL signal of Ru(bpy)32+ was almost inert towards ClO- (Figure S-6A) and PBS buffer with different pH (Figure S-6B). And ROS, RNS, glucose, AA, biothiols and common ions have nearly no effect on the FL signal of the m-CDs@Ru(bpy)32+ ratiometric sensors (Figure S-6C). These experimental results verified that Ru(bpy)32+ was one of the best reference reagent for ClOsensing. Figure S-6D shows that the zeta potential of m-CDs was as high as -24.7 mV and the zeta potential of Ru[(bpy)3]2+ was measured as +7.31 mV. These results indicate m-CDs and Ru[(bpy)3]2+ can form m-CDs@[Ru(bpy)3]2+ by electrostatical absorption. As shown in Figure 7A, when the concentration of the added ClO- in the mixed solution increased from 0 to 30 µM, the fluorescence intensity of m-CDs at 488 nm decreased gradually while that of Ru(bpy)32+ at 610 nm was stable. As a result, continuous photoluminescence color changes from cyan to red could be observed. In order to testify its utility for real sample assay, three different tap water sample were added to this ratiometric system. The corresponding color change showed that the ClO- concentration was 6-7 µM, and the outcome agreed with above-mentioned result of onefold m-CDs fluorescent nanoprobe (Figure 7B). These results illustrate mCDs@[Ru(bpy)3]2+ ratiometric fluorescent nanoprobe is applicable for semi-quantitative visualization detection in real water samples with high precision and practicability.

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Figure 7. (A) Under optimal conditions, fluorescent spectra of mixing m-CDs@[Ru(bpy)3]2+ ratiometric fluorescent nanoprobe for visual and specific detection of ClO-. The inset photos show the evolution of corresponding colors under 365 nm UV lamp (from right to left: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, and 30 µM ClO-, respectively). (B) m-CDs@[Ru(bpy)3]2+ ratiometric nanoprobe for the detection of ClO- in tap water samples.

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Luminescence Imaging of Exogenous ClO- in HeLa Cells. The cultured HeLa cells were incubated with the KRP buffer containing 20 µg•mL-1 m-CDs and m-CDs@[Ru(bpy)3]2+ for 1 h at 37 °C in a 5% CO2/95% air incubator, respectively. As shown in Figure 9A, the m-CDs-loaded cells only emitted strong background green fluorescence. After the cells were incubated with 10 µM NaOCl for another 15 min, the green fluorescence gradually weaken (Figure 9B). mCDs@[Ru(bpy)3]2+ treated with only PBS solution displayed intense red and green luminescence (Figure 9C), after the cells were incubated with 10 µM NaOCl for another 15 min showed a weaken fluorescence of green channel and a relatively stable fluorescence of red channel (Figure 9D). These results indicate that m-CDs and m-CDs@[Ru(bpy)3]2+ has been transferred into the cells and can react with the intracellular ClO- molecules.

Moreover, to simplify the detection of ClO- in real water samples, a portable test kit for the visual detection of ClO- was designed as a proof of concept by utilizing agarose hydrogel as a visual detection platform. Hydrogel is particularly suitable for designing the visual detection platform due to its negligible background color and fluorescence emission, as well as its large loading capacity and controllable shape.49 All components of the test kit, including polypropylene microcentrifuge tubes, agarose, m-CDs, Ru(bpy)3Cl2·6H2O, and PBS (0.1 M, pH = 7.0), are inexpensive, and the fabrication procedure was performed under ambient conditions. The portable test kit was applied to evaluate ClO- in the range of 0-200 µM. The detection procedure and the results were shown in Figure 8. The color of the hydrogel turned from cyan to red. Comparing with m-CDs agarose hydrogel (Figure S-7 shows the stability at various storage times), m-CDs@[Ru(bpy)3]2+ agarose hydrogel was colourful and more convenient to distinguish with the naked eye. Thus, the portable test kit is applicable for simple and convenient visual detection of ClO- in real water samples. Figure 9. Confocal fluorescence imaging of exogenous ClO- in HeLa cells. HeLa cells were first incubated with m-CDs (20 µg•mL-1) for 1 h at 37 °C and then were incubated with different solutions for 15 min: (A) PBS solution; (B) 10 µM NaClO. HeLa cells were incubated with m-CDs@[Ru(bpy)3]2+ (20 µg•mL-1 m-CDs and 40 µg•mL-1 [Ru(bpy)3]2+ mixed at v1/v2 = 6:1) for 1h at 37 °C and then were incubated with different solutions for 15 min: (C) PBS solution; (D) 10 µM NaClO. Fluorescence images were acquired using a confocal microscope with excitation and fluorescence emission windows of green channel (λex = 488 nm) and red channel (λex = 543nm).

Figure 8. (A) m-CDs@[Ru(bpy)3]2+-based agarose hydrogel solid matrix under visible light. (B) m-CDs-based agarose hydrogel solid matrix for the detection of ClO- under 365 nm UV lamp (from right to left: 0, 10, 20, 30, 40, 50, 60, 80, 100, and 200 µM ClO-, respectively). (C) m-CDs@[Ru(bpy)3]2+based agarose hydrogel solid matrix for the detection of ClOunder 365 nm UV lamp (from right to left: 0, 10, 20, 30, 40, 50, 100, and 200 µM ClO-, respectively).

CONCLUSIONS In summary, a facile one-step hydrothermal approach was adopted to prepare highly fluorescent carbon dots m-CDs with m-phenylenediamine as a precursor. Our investigation revealed that although the PLQY of m-CDs in ethanol was ~6.90%, which is similar to other CDs, its PLQY in water was as high as 31.58%. We also found that the strong fluorescence of m-CDs could be dramatically and specifically suppressed

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by ClO-. Further investigation revealed that differ from most of the previous approaches reported for the detection of ClO-, which were mainly based on the strong oxidizability of ClO-, the interaction between m-CDs and ClO- was most likely through the combination of N-H···O-Cl hydrogen bonding and energy transfer. Therefore, this dynamic quenching mechanism could well avoid the interference of other oxidizers, while these oxidizers could seriously interfere those ClO- detection methods based on the oxidation mechanism. Based on these findings, we developed a novel m-CDs@Ru(bpy)32+ ratiometric fluorescent nanoprobe for the visual detection of ClO-. The ratio and color change can be used for both qualitative recognition and quantitative analysis of ClO-. The linear response range of ClO- (R2 = 0.998) was from 0.05 to 7 µM and LOD was as low as 0.012 µM (S/N = 3). Moreover, a portable agarose hydrogel solid matrix-based mCDs@[Ru(bpy)3]2+ sensor has been developed for on-site detection of ClO- in water with the naked eye under a UV lamp, suggesting its potential in practical application with low cost and excellent performance in water quality monitoring. Additionally, intracellular detection of exogenous ClO- has been demonstrated via ratiometric imaging microscope, indicating the potential of the proposed ratiometric fluorescent nanoprobe for precise and reliable ratiometric imaging in living cells.

ASSOCIATED CONTENT Supporting Information. Determination of absolute quantum yields of mCDs; fluorescence spectrum, timeresolved fluorescence decay curves, PLQY, XPS of mCDs.; working mechanism of m-CDs for ClOsensing; fluorescence spectrum of [Ru(bpy)3]2+; the stability of mCDs agarose hydrogel with storage time; table (PDF)

AUTHOR INFORMATION Corresponding Author ∗

E-mail: [email protected] (LH Guo);

∗

E-mail: [email protected] (B Qiu).

Author Contributions 1 Zhan and Luo contributed equally to this work. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This project was financially supported by the Natural Science Foundation of China (21675028, 21575027, 21375021, and 21575025), Key Project of Fujian Province (2015Y0050), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R11).

ABBREVIATIONS ClO-, hypochlorite; CDs, carbon dots; PL, photoluminescence; QY, quantum yields; FL, fluorescence.

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