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Ratiometric Detection of Intracellular Lysine and pH with One-Pot Synthesized Dual Emissive Carbon Dots Wei Song, Wenxiu Duan, Yinghua Liu, Zhongju Ye, Yonglei Chen, Hongli Chen, Shengda Qi, Jiang Wu, Dan Liu, Lehui Xiao, Cuiling Ren, and Xingguo Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04211 • Publication Date (Web): 16 Nov 2017 Downloaded from http://pubs.acs.org on November 16, 2017

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Analytical Chemistry

Ratiometric Detection of Intracellular Lysine and pH with One−Pot Synthesized Dual Emissive Carbon Dots Wei Song,1 Wenxiu Duan,2 Yinghua Liu,1 Zhongju Ye,3 Yonglei Chen,1 Hongli Chen,1 Shengda Qi,1 Jiang Wu,1 Dan Liu,2 Lehui Xiao,3,* Cuiling Ren,1,* and Xingguo Chen1 1

State Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, P. R. China.

2

School of Life Sciences, University of Science and Technology of China, Hefei, 230027, P. R. China.

3

College of Chemistry, Nankai University, Tianjin, 300071, P. R. China.

1 Environment ACS Paragon Plus


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ABSTRACT Recently, the development of new fluorescent probes for the ratiometric detection of target objects inside living cells has received great attention. Normally, the preparation, modification as well as conjugation procedures of these probes are complicated. On this basis, great efforts have been paid to establish convenient method for the preparation of dual emissive nanosensor. In this work, a functional dual emissive carbon dots (dCDs) was prepared by a one−pot hydrothermal carbonization method. The dCDs exhibits two distinctive fluorescence emission peaks at 440 and 624 nm with the excitation at 380 nm. Different from the commonly reported dCDs, this probe exhibited an interesting wavelength dependent dual responsive functionality toward lysine (440 nm) and pH (624 nm), enabling the ratiometric detection of these two targets. The quantitative analysis displayed that a linear range of 0.5−260 µM with a detection limit of 94 nM toward lysine and the differentiation of pH variation from 1.5 to 5.0 could be readily realized in a ratiometric strategy, which was not reported before with other carbon dots (CDs) as the probe. Furthermore, because of the low cytotoxicity, good optical and colloidal stability, and excellent wavelength dependent sensitivity and selectivity toward lysine and pH, this probe was successfully applied to monitor the dynamic variation of lysine and pH in cellular systems, demonstrating the promising applicability for biosensing in the future.

Keywords: Carbon dots; Ratiometric; Intracellular imaging; Lysine; pH


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INTRODUCTION Fluorescent ratiometric sensing is based on the detection of the ratio of fluorescence (FL) intensities under two well resolved wavelengths, which can effectively eliminate the influence induced by the fluctuation of excitation source, local environment change, and variation of the probe concentration. The detection accuracy can therefore be greatly improved. Recently, the development of new fluorescent probes for the simultaneous ratiometric differentiation of two different targets from dual channels has received more attention because this sensing strategy can provide further detection information for biological or biomedical analysis. So far, some interesting works have been reported.1−4 For example, Huang et al. developed a nanosensor for ratiometrical detection of O2•− and pH by conjugating hydroethidine, fluorescein isothiocyanate, and (4−carboxybutyl) triphenylphosphonium bromide onto the silica coated CdSe/ZnS quantum dots.1 Chen et al. have designed and synthesized a dual−detection organic fluorescent probe to detect H2S and H2Sn with distinctive fluorescence signals.2 Recently, Zhou and coworkers have prepared a two−photon fluorescence probe for ratiometric visualization of NO/H2S.3 However, the majority of these designs are typically involved in complicated preparation, modification or conjugation procedures. On this basis, the establishment of convenient yet robust method for the preparation of dual emissive nanosensor is fundamentally significant. Until now, the commonly adopted ratiometric sensors are usually designed by combining two components with distinct emission wavelength together. Examples include single emission carbon dots (CDs),5−7 fluorescent molecules,8−10 fluorescent proteins,11 polymer dots,12,

13

upconversion nanoprobes,14 quantum dots,15 metal cluster,16 metal−organic framework,17 and so on.18, 19 Furthermore, the dual emissive organic molecules,20−24 Mn doped quantum dots,25, 26 metal−organic framework,17, 27 and CDs28, 29 can realize ratiometric detection by itself. Among



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these four kinds of dual emissive fluorescent materials, carbon nanoparticles received more attention for the construction of ratiometric fluorescent sensor because of the unique optical properties, excellent biocompatibility, good water solubility, and convenient surface modification flexibility.30, 31 To the best of our knowledge, even though a few dual emissive CDs have been reported before,28,

29

the method to generate CDs with wavelength−resolved dual

target sensing capability remains rare. In this work, a new wavelength−resolved multi−targets responsive dCDs was prepared by a one−pot hydrothermal method. As illustrated in Scheme 1, the procedure to fabricate the dCDs is very convenient, i.e., one−pot hydrothermal carbonization. No further post surface modification or conjugation was required. Moreover, this dual emissive sensor can be excited by single−wavelength, which could effectively avoid the complicated instrumental operation, the interference of background noise and auto−fluorescence from cells.32 Most importantly, this probe can response to pH and lysine by distinctive emission wavelength, which is more favorable for real sample analysis. The sensitive and selective detection of lysine was significant for diagnosing various disease and disorders because a high concentration of lysine in plasma and urine is an indicator of congenital metabolic disorders.33 Meanwhile, pH measurement in acidic conditions in living systems is necessary and meaningful because acidic pH plays a fundamental role in many organelles of eukaryotic cells, especially those organelles along the secretory and endocytic pathways.34 Interestingly, the as-synthesized dCDs can complete the two tasks in a ratiometric manner. To the best of our knowledge, this is the first report of dCDs for ratiometric detection of two targets under distinctive emission wavelengths. As a consequence, the unique features as noted above together with the good water dispersibility, optical stability as


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well as cell biocompatibility would enable this new sensor find broad applications in biosensing, particularly in cellular system in the future.

Scheme 1. The schematic diagram of the preparation procedure of dCDs and specific ratiometric detection of lysine and pH.

EXPERIMENTAL SECTION Preparation of Dual Emissive Carbon Dots (dCDs). The dCDs were prepared by a one−pot hydrothermal carbonization method. Briefly, 0.4477 g of o−phenylenediamine (oPD) was mixed with 15 mL of phosphoric acid within a Teflon−lined autoclave and then heated at 200 ℃ for 24 h. After the autoclave was cooled down to room temperature naturally, the dark blue product was centrifuged at 11000 rpm for 30 min to remove large particles and the supernatant was collected. The control experiments were conducted identically except phosphoric acid was replaced by other control solution. For example, oCDs was prepared by 0.4477 g of oPD and 15 mL of H2O. The mixture of oCDs with phosphoric acid solution was prepared by adding 0.4477 g oCDs into



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15 mL of phosphoric acid and standing at room temperature for 24 h. The FL spectra were measured and the intensity of FL peak at 440 and 624 nm was noted as F440 and F624, respectively. Detection of Lysine and pH by dCDs. For the detection of lysine in solution, 10 µL of lysine with different concentrations were separately added into the mixture of 3.0 mL buffer solution (citric acid–Na2HPO4, 100 mM, pH=2.0) containing the as-synthesized dCDs. If there is no special statement, the final concentration of dCDs is 2.8 µg/mL. The selectivity of the ratiometric nanosensor towards lysine was evaluated by adding 10 µL of other metal ions, amino acid or glutathione solutions instead of lysine in a similar way. For the detection of pH, the prepared dCDs were added into 3.0 mL of buffer solution with different pH (citric acid–Na2HPO4, 100 mM) and the FL spectra were recorded. To explore the feasibility of dCDs for lysine assay in biological samples, human serum samples were obtained from the first affiliated hospital of Lanzhou University. The authors state that all experiments were performed in compliance with the relevant laws and institutional guidelines. The serum samples were subjected to a 100-fold dilution before analysis. Diluted samples were spiked with various concentrations of standard lysine solution. All of the FL spectra were recorded under the excitation at 380 nm and all experiments were performed at room temperature. Cell Imaging. For live cell imaging, HeLa cells were plated on 18×18-mm glass coverslips mounted in custom-designed chambers and cultured in L-15 medium without phenol red (Invitrogen).


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Temperature was maintained at 37 ℃ using an air stream incubator. HeLa cells were incubated with 100 µg/mL dCDs for 6 h at 37 ℃. Then the cells were washed with PBS buffer after removing the cell culture medium and the images were acquired with a Nikon inverted Eclipse Ti microscope with a 100×1.40 NA objective. Cells were excited at 405 nm and emissions were acquired separately with two different single band fluorescence filters corresponding to the red (580−653 nm) and blue (467−499 nm) fluorescence. To test the pH sensing capability, the dCDs was added into E. coli solution and incubated for 2 h at 37 ℃. Then the sample was centrifuged to remove additional dCDs in the cell culture medium. The dCDs loaded E. coli was then redispersed in the buffer with different pH (citric acid–Na2HPO4, i.e., 2.5 to 7.4) for 1 h. The samples were imaged under the same setup as noted above.

RESULTS AND DISCUSSION Microscopic and Spectroscopic Characterization of dCDs. The morphology and size of the as-synthesized dCDs were characterized by TEM. As shown in Figure 1A, the particles are nearly spherical with an average diameter of ~ 5 nm. Their size distribution is narrow according to the statistically analyzed results from more than 100 nanoparticles (insets of Figure 1A), while the particles showed no obvious crystal lattice, suggesting an amorphous structure. The XRD pattern (Figure 1B) of dCDs further confirmed this argument.30 The photoluminescence property of dCDs was investigated by FL spectra. As shown in Figure 1C, the prepared dCDs show two emission peaks at around 440 and 624 nm, and their emission intensity was all influenced by excitation wavelength. Take into account the emission intensity at the two wavelengths, 380 nm was chosen as the excitation wavelength in



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the following experiments. The fluorescence lifetime of the prepared dual emissive carbon nanoparticles is about 5.0 ns.

Figure 1. TEM image (A), XRD pattern (B) and FL spectra under different excitation wavelengths (C) of dCDs. FTIR spectra (D) and High resolution XPS spectra of C1s (E), N1s (F) and P2p (G) of the as-synthesized dCDs.

The surface functional groups of dCDs were identified by FTIR spectroscopy. As shown in Figure 1D, the broad absorption bands at 3120-3650 cm−1 are attributed to the stretching vibrations of O−H and N–H.31, 35 The band at 2920−3030 cm−1 is the characteristic absorption of CH2.36 The absorption bands at 1356−1790 cm−1 is assigned to the stretching vibrations of C–N, C=C, and/or C=O.37,

38

The stretching vibrations of P=O and P−O−R (R= alkyl group) are

located at 940−1260 cm−1.28, 36 In addition, the bending vibration of C–H is corresponding to the absorption bands at 680−890 cm−1.28 These observations suggest that the prepared dCDs were mainly composed of C, O, N and P. Additionally, the FTIR spectra of dCDs is noticeably


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different from that of oPD and phosphoric acid, demonstrating the successful hydrothermal carbonization process of oPD and phosphoric acid. The valence states of the elements within the dCDs were analyzed by XPS. The full range XPS spectrum (Figure S1) further confirmed that the elemental composition of dCDs are C, O, N and P, in good agreement with the FTIR spectroscopic measurement. The high resolution C1s spectrum (Figure 1E) could be de-convoluted into four peaks, at 284.5 eV (C=C), 285.1 eV (C−N/C−P=O), 286.0 eV (C=O), and 288.0 eV (O−C=O).36, 39 The N1s spectrum (Figure 1F) shows three peaks at 398.7, 400.0 and 401.0 eV, which can be assigned to pyridinic N, pyrrolic N and N−H, respectively.40,

41

The P2p spectrum (Figure 1G) reveals the presence of P=O

(133.1 eV) and P−C/P−N (134.1 eV) groups.39 Taken together, the FTIR and XPS characterizations illustrated the successful incorporation of phosphorus and nitrogen into the dCDs. In order to understand the formation mechanism of dCDs, further control experiments were conducted.

For

example,

oCDs

were

prepared

by

hydrothermal

carbonization

of

o−phenylenediamine (oPD) in H2O. Comparable FL (Figure 2A) and UV−Vis absorption (Figure 2B) spectra from oCDs and their mixture with phosphoric acid were observed, which are significantly different from that of dCDs. These results demonstrated that dCDs were not the simple mixture of oCDs and phosphoric acid. The UV−Vis absorption band of oCDs and their mixture with phosphoric acid was mainly located at 190−320 nm, which are corresponding to π−π* transitions of C=C bonds and n−π* transitions of C=O bonds.35, 38 Distinct from oCDs, dCDs exhibit new adsorption peaks in the range of 500−650 nm, which might contribute to the FL emission in red wavelength.42



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Figure 2. FL (A) and UV−Vis absorption (B) spectra for oCDs (blue line), their mixture with phosphoric acid (black line) and dCDs (red line).

According to the FL spectra of dCDs and oCDs, it is evident that phosphoric acid played an important role in controlling the fluorescent properties of dCDs. In order to figure out whether the effect of phosphoric acid is ascribed to the phosphorus atom doping or acid property, further control CDs were prepared and their FL spectra are illustrated in Figure S2a. It is noticeable that the CDs prepared by the mixture of oPD and trisodium phosphate or oPD and ammonium phosphate all showed two emission peaks at short wavelength, suggesting phosphorus precursor played a key role in controlling the dual emission property of dCDs. Interestingly, the product of CDs from the mixture of oPD and citric acid solution only showed one emission peak at short wavelength, indicating a minor effect of dilute acid on regulating the FL properties of dCDs. However the product of CDs prepared by oPD and concentrated sulfuric acid showed one emission peak at long wavelength. From the UV−Vis absorption spectroscopic measurements, the CDs synthesized either in weak or strong acid environment were all different from that of dCDs (Figure S2b). As a consequence, these results suggest that the concentrated acid properties of phosphoric acid also played an important role in the formation of red emission dCDs.


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To the best of our knowledge, some groups have reported nitrogen and phosphorus co−doped carbon nanoparticles before.28, 43, 44 These CDs either showed one or two emission peaks at short wavelength. From these results, we speculated that the chemical structure of the precursor should play an important role in controlling the FL property of dCDs. In order to ascertain this argument, two isomers of oPD (m−phenylenediamine and p−phenylenediamine) were also used to prepare CDs under the same reaction condition as that of dCDs. The FL spectra were shown in Figure S2c. It is evident that those two products showed only one emission peak at 430 and 510 nm, respectively. The UV−Vis absorption spectra were also different from that of dCDs (Figure S2c). Taken together, these results demonstrated that the successful formation of dCDs is the combined effect from concentrated acid, doped nitrogen and phosphorus and the structure of o−phenylenediamine. Quantifying Lysine with dCDs in a Ratiometric “Turn−On” Mode at Blue Wavelength. Lysine is bound up with the Krebs–Henseleit cycle and polyamine synthesis.45 High concentration in plasma and urine is an indicator of congenital metabolic disorders.33 As a consequence, the sensitive and selective detection of lysine was meaningful for diagnosing various disease and disorders. Interestingly, we found that the FL intensity of dCDs at 440 nm could be enhanced by lysine, while the peak at 624 nm was kept constant (Figure 3A), affording a robust ratiometric sensor in a “turn−on” mode. The inserted figures in Figure 3A displayed the solution color change under UV light irradiation, i.e., from pink to purple after the addition of lysine gradually. In order to achieve the optimum detection performance, the effect of pH on the detection of lysine was studied (Figure S3 and Figure 3B). The results indicated that the as-synthesized dCDs could be used to ratiometrically detect lysine in the pH range of 1.5−5.0 and it could also



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be used as a single wavelength sensor for lysine assay at pH 7.4. The detection linear range and the detection limit were listed in Table S1. In order to realize the lowest detection limit and the widest linear range, pH 2.0 was chosen as the optimal pH for lysine detection. Under this condition, a linear relationship can be obtained in the concentration range of 0.5−260 µM, and detection limit can be reached to 94 nM. This detection process can be completed within 2 min (Figure S4).

Figure 3. (A) FL spectra of dCDs after adding different concentration of lysine (0.5–260 µM) in 100 mM buffer solution at pH 2.0. (B) Linear relationship between F440/F624 and the concentration of lysine. F440/F624 variation (C) and FL spectra (D) of dCDs in the presence of different amino acids and metal ions (concentrations of interferences are 0.78 mM except glutathione is 10 mM, and the concentration of lysine is 0.26 mM).

The selectivity of this sensor was evaluated by parallel measuring the FL response of dCDs to other amino acids, glutathione and some metal cations. As shown in Figure 3C and 3D, the


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influence of these interferents on the detection performance of this sensor towards lysine is negligible. While FL spectra indicated the emission at 624 nm was inert to all these interferents and lysine. These results declare that dCDs can realize selective ratiometric detection of lysine. The high sensitivity and selectivity of dCDs make it a promising candidate for the detection of lysine in real samples. The detection results of lysine in human serum are listed in Table S2. The recovery ranged from 97.9% to 103.2%, demonstrating a high accuracy for real sample analysis. To understand the sensing mechanism of dCDs to lysine, TEM, FTIR, FL, UV−Vis absorption spectra and zeta potential before and after the addition of lysine were measured. As shown in Figure S5a, the FL intensity of dCDs at 440 nm can be greatly enhanced by lysine, but the particle size and colloidal stability showed no evident change (Figure S5b and S5c), suggesting that the FL variation was not stemming from the particle state variation. As far as we know, the FL property of the CDs can be enhanced by protein,46 organic species, polymer or silica through surface passivation,47 lysine can also passivate the surface of BSA coated CDs and enhance their FL intensity.46 Therefore, judging from the above mentioned results, the FL enhancement by lysine was probably due to surface passivation. FTIR spectra of dCDs before and after the addition of lysine were recorded (Figure S5d), their main infrared absorption peaks showed no obvious difference. UV−Vis absorption spectroscopy was further used to certify this inference. As shown in Figure S5e, the absorbance in the range of 250 to 400 nm was increased upon the addition of lysine, which can prove the occurrence of passivation preliminary.48 Furthermore, the zeta potential of the prepared dCDs was 4.23 mv (Table S3), proving that amino groups were in majority. The present of lysine initiates the decrease of the zeta potential to 1.37 mV, which can be explained that lysine can partially shield the positively charged amino groups of dCDs. This result further proves that surface passivation was occurred.49 In order to understand whether the



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selective sensing of dCDs towards lysine came from the amino or carboxy group of lysine, the fluorescence response of dCDs towards 1, 5−diaminopentane, 6−aminocaproic acid and DL−norleucine, which lack one α−COOH, α−NH2 and ε−NH2 than lysine, respectively, were tested. As shown in Figure S6, the emission peak at 440 nm has no response to all of the three chemicals, implying all of the amino and carboxyl functional groups of lysine played important roles in the detection process. Ratiometric Measurement of pH with dCDs in a “Turn−off” Mode at Red Wavelength. Monitoring pH changes in live cells is crucial for the better understanding of physiological and pathological processes. Many pH sensors have been developed,9,

13, 29, 32

Unfortunately, little

extreme acidity pH (pH < 4.0) probes have been reported.34, 50 Though strong acidic environment is fatal for the majority of living organisms, a considerable number of microorganisms such as acidophiles and helicobacter pylori can still live under such harsh conditions. Moreover, enteric bacteria such as Salmonella species and E. coli can survive through the highly acidic mammalian stomach. Therefore, the development of new fluorescent probes which can be used in extreme acidic conditions in cellular systems is necessary and meaningful. From the spectroscopic measurements, the FL signal of dCDs at 624 nm is sensitive to pH in the range of 1.5−5.0, while the FL intensity at 440 nm was inert to it (Figure 4A). The color of the solution was gradually changed from blue to pink and the red FL emission under UV light irradiation was gradually weakened with the pH increasing (insets of Figure 4A). Therefore, this sensor can be used for ratiometric pH sensing under extreme acidic condition. As expected, the value of F624/F440 was in a linear relationship with pH in the range of 1.5−5.0 (Figure 4B). Notably, no significant change of the F624/F440 value after repeated three times measurements (inset of Figure 4B) was noted, declaring a reversible and stable sensor for pH assay.


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Figure 4. (A) FL emission spectra of the as-synthesized dCDs at different pH (from 1.5 to 5.0) buffer solution (insets are the corresponding solutions under white light (top) and UV light (down) irradiation. From left to right, pH is from 1.5 to 5.0). (B) Linear relationship between F624/F440 and pH, inset is the reversible response of F624/F440 towards pH in the range of 1.5−5.0.

According to the previous work, the excitation−dependent photoluminescent behavior is attributed to the surface state which can affect the band gap of CDs.30,

51, 52

The excitation

independent property of the emission peak at 624 nm might stem from the uniform sp2 domains of the dCDs.53 Therefore, we conclude that the red emission might stem from the sp2 domain in the particle, while the shorter emission wavelength was related to the surface or edge functional group. It is inferred that the response of dCDs at longer wavelength towards pH may stem from protonation and deprotonation of the doped N in the rigid carbon skeleton structure.54 In acid condition, the edge/surface groups and the nitrogen in the rigid carbon skeleton were all protonated. When the pH of the solution increased, the doped nitrogen in dCDs was first deprotonation which influences the sp2 domains of the dCDs. Therefore, the FL intensity at 624 nm was quenched. At the same time, the edge/surface groups were not changed in this process,



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so the FL intensity at 440 nm was kept constant in the pH range of 1.5−5.0. In order to prove this speculation, FL spectra, FTIR spectra and zeta potential of dCDs at different pH were recorded. As expected, the FL intensity at 624 nm was nearly quenched completely at pH 6.0 (Figure S7a), while the FL intensity at 440 nm was not changed, but this value was suddenly decreased at pH 6.0, suggesting the edge/surface groups were deprotonation. As shown in Figure S7b, the absorption bands at 1356−1790 cm−1 was changed at some extent with the pH increasing, proving that the pH response of dCDs stems from the protonation and deprotonation of the C−N.55 In addition, the zeta potential of the prepared dCDs became more negative with the pH increasing (Table S3), further confirming the deprotonation process. Imaging of Lysine and pH in Living Cell Systems. Owing to the excellent specificity as well as good sensitivity of dCDs toward lysine and pH measurement in solution, it should be very interesting to explore the capability of this probe in biological sample assay. Before understanding the applicability of dCDs in intracellular imaging, the cytotoxicity to living cells was evaluated by MTT assay. Figure S8 depicts the viability of HeLa cells after treated with various concentrations of dCDs from 50 to 400 µg/mL. More than 89% of HeLa cells were viable even at the dosage of 400 µg/mL, indicating the potential of the probe in intracellular imaging of living cells. To explore the fluorescence stability of these dCDs for biological imaging, we illuminated the sample in the buffer solution with different concentration of NaCl or after irradiation by UV light for 1 h, respectively. The FL intensities from the dCDs at 440 and 624 nm were kept constant as shown in Figure S9a and S9b. Moreover, increasing the temperature of the solution from 10 to 50 ℃ still did not affect the FL property of dCDs (Figure S9c). The excellent photo-physical


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stability of the prepared dCDs makes them hold great potential in biological applications. The confocal images of HeLa cells (Figure S10) treated with dCDs display their good cell imaging capability in blue and red channels. HeLa cells and E. coli were chosen as model organisms for evaluating the potential of dCDs in cell imaging of lysine and pH, respectively. As shown in Figure 5A, the fluorescent intensity of HeLa cells incubated with dCDs in the red channel was almost keep constant in the presents of different concentration of lysine, meanwhile, the emission intensity in the blue channels was increased gradually when the concentration of lysine changed from 0 to 1000 µM, proving that the prepared dCDs can be used to image the concentration variation of lysine in HeLa cell. In order to determine whether dCDs work in bacterial cells under such acidic conditions or not, E. coli was incubated with dCDs in the culture medium for 2 h. Then E. coli was re-dispersed in the buffer with different pH (citric acid–Na2HPO4, i.e., 2.5 to 7.4) for 1 h. The samples were imaged under the same setup as noted above. As shown in Figure 5B, the prepared dCDs can be used to label E. coli in blue and red channels. The fluorescence intensity of E. coli incubated at pH 2.5 was noticeably higher than that at pH 7.4 in red channels, while the fluorescence intensity was almost the same in blue channels, suggesting the prepared dCDs can be used to image the pH variation inside E. coli.



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Figure 5. (A) Representative fluorescence images of HeLa cells treated with different concentrations of lysine in red and blue channels. HeLa cells were incubated with dCDs (100 µg/mL) for 3 h with different concentration of lysine (0, 500 and 1000 µM). Scale bar is 50 µm. (B) Visualization of pH changes in E. coli by confocal laser scanning microscopy at pH 2.5 and 7.4. The excitation wavelengths were 405 nm. Emission was collected in the blue and red channels. Scale bar, 10 µm.

CONCLUSIONS In conclusion, a new dCDs was prepared by a simple one−pot hydrothermal carbonization method. The comprehensive spectroscopic characterizations demonstrated that this new fluorescent probe can be used for the differentiation of lysine and pH in a ratiometric method from two separated wavelengths i.e., 440 and 624 nm. A detection linear range of 0.5−260 µM with a detection limit of 94 nM toward lysine and pH variation between 1.5 and 5.0 were readily


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realized. By the virtue of good stability and high biocompability, this sensor could be applied to ratiometricly monitor the concentration variation of lysine or pH change inside living cells, indicating its potential practical applications for the diagnosis of lysine and pH related disease and disorder.

ASSOCIATED CONTENT Supporting Information Detailed information about materials, characterization and instrumentation, MTT assay, supplementary figures and tables. These materials are available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION *Corresponding Author Fax: +86/931/8912582. E-mail: [email protected]; [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We are grateful for the financial supported by the National Natural Science Foundation of China (No. 21207057, 21522502) and the Fundamental Research Funds for the Central Universities (lzujbky-2016-43).

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