Terbium (III) modified fluorescent carbon dots for highly selective and

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Terbium (III) modified fluorescent carbon dots for highly selective and sensitive ratiometry of stringent Bin Bin Chen, Meng Li Liu, Lei Zhan, Chun Mei Li, and Chengzhi Huang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05149 • Publication Date (Web): 01 Mar 2018 Downloaded from http://pubs.acs.org on March 1, 2018

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Analytical Chemistry

Terbium (III) modified fluorescent carbon dots for highly selective and sensitive ratiometry of stringent Bin Bin Chen,a Meng Li Liu,a Lei Zhan,b Chun Mei Li*b and Cheng Zhi Huang*ab a.

Key Laboratory on Luminescence and Real-Time Analytical Chemistry, Ministry of Education College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400716, China.

b.

Chongqing Key Laboratory of Biomedical Analysis (Southwest University), Chongqing

Science & Technology Commission, College of Pharmaceutical Science, Southwest University, Chongqing 400716, China. *

Corresponding author. Tel: (+86)-23-68254659. E-mail addresses: [email protected]; [email protected].

ABSTRACT: Highly selective and sensitive detection of guanosine 3’-diphosphate-5’diphosphate (ppGpp), namely the stringent in plants or microorganisms responding to strict or extreme environmental conditions such as stress and starvation, which plays an important role in gene expression, rRNA and antibiotics production, regulations of virulence of bacteria and growth of plants, faces a great challenge owing to its extreme similarity to normal nucleotides. By modifying the surface groups of a facile two-step hydrothermal route prepared carbon dots (CDs) with terbium ions (Tb3+) in this contribution, a novel fluorescent probe with excellent properties such as highly physical and chemical stability, narrow emission and excitation wavelength-independent emission was prepared. The Tb3+ ions on the surface of CDs can not only preserve the intrinsic fluorescence (FL) of CDs, but also keep its own coordination capacity with rare earth complex, and thus the clamp structure (four phosphate groups) of ppGpp can

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specific binding with Tb3+ ions on the surface of CDs to produce antenna effect. Therefore, a highly selective and sensitive fluorescent ratiometry of ppGpp was developed by terbiummodified carbon dots (CDs-Tb) with the limit of detection as low as 50 nM based on the synergistic effect of antenna effect of Tb3+ ions and specific recognition capacity of CDs. The applicability of this assay was demonstrated by CDs-Tb-based paper sensor for high distinguishing ppGpp from other nucleotides with similar structure. KEYWORDS: Terbium-modified carbon dots, fluorescent ratiometry, ppGpp INTRODUCTION Guanosine 3’-diphosphate-5’-diphosphate (ppGpp) is an effector molecule of stringent response that generates in bacteria and plants under stress and starvation conditions by RelA and SpoT pathways, and plays an important role in gene expression, rRNA and antibiotics production and regulating virulence.1-3 Owing to the high structural similarity of ppGpp to other nucleotides (such as GTP and GDP), selective and sensitive detection of ppGpp is always an annoying problem, hampering its further study, particularly the real-time or on-size monitoring. Although a variety of detection methods of ppGpp, including high-performance liquid chromatography (HPLC)4-5, thin layer chromatography (TLC)6 and conventional fluorescence (FL) methods7-9, have been developed, but they suffer from mediocre sensitivity and selectivity, lengthy procedures, or necessity for expensive equipment and high skills. Therefore, developing a simple, highly sensitive and selective detection method of ppGpp is very important in terms of the stringent response of bacteria and plants in the conditions of stress and starvation, which not only can help to understand the RelA and SpoT pathways, but also the developments of new biomedicines. Fluorescent carbon dots (CDs), developed as a smart family of fluorescent nanomaterials, have a core-shell structure with the nanocrystalline core of sp2-hybridized carbon clusters and the functional group formed shell of sp3-hybridized carbons.10-11 In


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comparison to semiconductor quantum dots and FL dyes, the fluorescent CDs are proudly characterized by good biocompatibility, tunable emissions, excellent photostability and catalytic activity, and therefore, have been widely applied in cellular imaging, biochemical sensing and organocatalysis.10,

12-21

Currently, the element doping and

surface modification are two main functionality approaches, which enable the chemical, optical, and electrical properties of CDs tunable and controllable.22 Surface modification can greatly change the surface state of CDs by introducing functional groups with biological molecules and small molecules, such as L-cysteine23, organosilane24, tyramine25, polyethylenimine (PEI) and nuclear localization signal (NLS) peptide26-27, etc. The metal ions, especially the rare earth ions, are rarely employed to modify the surface of CDs.11, 2829

Although doping metal ions, such as terbium-doped CDs (Tb-CDs) and europium-doped CDs

(Eu-CDs) by one-pot carbonization synthesis, can efficiently change the electronic structure so as to create n-type and p-type carriers, and can significantly improves the stability and oxidation resistance of CDs,11, 28 it is difficult to keep origin properties of metal ions. For example, the antenna effect, which is the process of light absorption, energy transfer and emission, disappears because rare earth ions doped in CDs is very difficult to combine with complexes.11, 28 Rare earth ions have low luminescent efficiency, but can emit strong FL owing to the antenna effect when specifically integrating with ligands. So, it is desirous if the coordination capacity of rare earth ions could be preserved during the surface modification. However, the preparation of rare earth ions modified CDs faces many challenges such as difficulties in modification and easy aggregation. Therefore, it still remains highly desirable to develop a simple rapid way for preparation of rare earth ionsmodified CDs. In this work, we prepared terbium-modified fluorescent CDs (CDs-Tb) through a twostep preparation route, wherein the blue-emissive CDs are firstly prepared by hydrothermal route, and then Tb3+ ions are coordinated with carboxyl and amine groups


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on the surface of CDs to form CDs-Tb (Scheme 1). The narrow emission peak of the CDs in aqueous solution remains unchanged and thus could serve as a reference signal, while the Tb3+ ions exhibit enhanced sharp FL emission signal upon binding with ppGpp and thus serve as the response signal (Scheme 1). The fluorescent ratiometry not only improves the detection sensitivity but also avoids interference from the background FL.3031

Thus reference signal and response signal enable a highly sensitive ratiometry of

ppGpp with the limit of detection at nanomolar level, which is the lowest value reported up to now.7 Importantly, the general nucleotides and metal ions scarcely exert influence on the FL emission of CDs-Tb, indicating that the proposed ratiometry of ppGpp is highly selective in the complex samples.

Scheme 1. Illustration of the synthesis process of CDs-Tb for highly selective detection of ppGpp, in which CDs are first prepared from citric acid and triethylenetetramine (TETA) by the hydrothermal method.

EXPERIMENTAL SECTION Reagents Adenosine 5’-triphosphate disodium salt hydrate (ATP), uridine 5’-triphosphate trisodium salt hydrate (UTP), cytidine 5’-triphosphate disodium salt (CTP), guanosine 5’-triphosphate sodium salt hydrate (GTP), guanosine 5’-diphosphate sodium salt (GDP), guanosine 5’-monophosphate disodium salt hydrate (GMP) and citric acid were all purchased from Sigma Aldrich. The ppGpp was obtained from Trilink (USA). Terbium (III) nitrate pentahydrate (Tb(NO3)3·5H2O) and


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triethylenetetramine (TETA) were commercially available from Aladdin Reagent. Sodium phosphate tribasic (Na3PO4·12H2O) and phosphoric acid (Pi) were both from Chuandong Chemical Group Co., Ltd (Chongqing, China). Sodium pyrophosphate (PPi) was from Beibei Chemical Reagent Factory (Chongqing, China). Instruments The UV absorption spectrum of CDs was obtained from a Hitachi U-3010 spectrophotometer (Tokyo, Japan). The elemental compositions of CDs and CDs-Tb were measured with an ESCALAB 250 X-ray photoelectron spectroscopy. The FT-IR spectra of CDs and CDs-Tb were collected on a Hitachi 8400S Fourier Transform Infrared spectrometer (Tokyo, Japan). The TEM and HRTEM data of CDs and CDs-Tb were performed on a Tecnai G2 F20 field emission transmission electron microscope (FEI, USA). The FL spectra of CDs and CDs-Tb were recorded with a Hitachi F-2500 FL spectrophotometer (Tokyo, Japan). Zeta potentials of CDs and CDs-Tb were measured using a Zetasizer Nano-ZS System (Malvern, English). The FL lifetimes of CDs and CDs-Tb were measured with a FL-TCSPC FL spectrophotometer (Horiba Jobin Yvon, France). Preparation and purification of CDs Citric acid (100 mg) and TETA (200 µL) were dissolved in 5.0 mL water, and the obtained solution was transferred into a 25 mL Teflonlined stainless steel autoclave and heated at 160 oC for 3 h. Then the autoclave was cooled down naturally and subjected to dialysis process. Through a cellulose ester dialysis membrane (500-1000 MWCO), residual amounts of citric acid and TETA were removed over 24 h. The resulting material was dried by lyophilization to obtain CDs, which was dispersed in water for further characterization and use. Surface modification of CDs with Tb3+ Aqueous CDs (10 mL, 0.3 mg/mL) solution was mixed with 5 mL of aqueous Tb(NO3)3 solution (0.1 M) for 3 h at room temperature. The resulting mixture was then dialyzed with a cellulose


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ester dialysis membrane (500-1000 MWCO) for 4 days to remove “free” Tb3+ ions. The resulting material was dried by lyophilization to obtain CDs-Tb, which was dispersed in water (0.5 mg/mL) for further characterization and use. Binding of CDs-Tb with ppGpp and other nucleotides Titration of CDs-Tb with ppGpp was made by adding various concentrations of ppGpp (0-25 µM) into the CDs-Tb solution (50 µg/mL in Tris-HCl buffer, pH=6.52), and then subjected to FL measurements after 5 min. The selectivity for ppGpp was confirmed by adding other nucleotides stock solutions instead of ppGpp in a similar way at a concentration of 15 µM. All experiments were performed at room temperature. RESULTS AND DISCUSSION Characteristics of the CDs and CDs-Tb The blue-emissive CDs can be facilely prepared by hydrothermal route using citric acid and TETA. The TEM image (Figure 1a) shows that the formed CDs are well monodispersed with average diameter of 7.5 ± 3.5 nm (Figure 1a, inset). The HRTEM image of CDs (Figure S1, SI) clearly shows the lattice spacing of 0.21 nm, which is corresponding to the (020) diffraction planes of graphite.10 The XPS (Figure 1b, blue curve) is used to explore surface elements and chemical bonds of CDs. Three peaks at 285.6, 401.3 and 532 eV are attributed to C1s, N1s and O1s, respectively.10, 32 The high resolution XPS spectra of C1s, N1s and O1s (Figure S2, SI) reveal the presence of C-C, C-N, C=N/C=O and N-H bonds in CDs. The Fourier transform infrared (FT-IR) spectrum (Figure 1c, black curve) of CDs exhibits the νC-H bands in the 2851 and 2920 cm-1 regions, as well as the νO-H, νC=O, δN-H, νC-N and νC-O bands at 3426, 1641, 1566, 1385 and 1111 cm-1,10, 16 which is basically consistent with the results of XPS and further confirm the presence of hydroxyl, amide, amino and carboxyl groups.


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Figure 1 The morphology, elemental analysis, FL properties and zeta potentials of the CDs. (a) TEM image of CDs (Inset: particle size distribution ); (b) XPS spectra of CDs and CDs-Tb; (c) FT-IR spectra of CDs and CDs-Tb; (d) absorption and FL spectra of CDs (Inset: photograph of CDs solution under the excitation of the 365 nm UV light lamp). (e) FL lifetimes of CDs and CDs-Tb; (f) zeta potentials of CDs and CDs-Tb.

Owing to the π-π* transition of C=C and n-π* transition of C=O, the CDs display two strong absorption bands characterized at 240 nm and 354 nm, respectively (Figure 1d).33 Under the excitation of 310 to 390 nm light beam, the CDs show excitation wavelengthindependent FL emissions with a narrow FL band, and the maximum emission can be available at 463 nm with the maximum excitation at 360 nm (Figure 1d). Under the 365 nm UV light lamp, the CDs solution shows a brightly blue FL emission (Figure 1d, inset). The relative quantum yield of the CDs is up to 54.8% (quinine sulfate as reference, Figure S3, SI), which is beneficial for application such as in cellular imaging and analytical field. Importantly, the CDs own great stabilities under continuous illumination for 150 min (Figure S4, SI) or in a high ionic environment of 4 M NaCl solution (Figure S5, SI), indicating that CDs have great potential toward designing FL probe for the analytical detection in the complex samples. Moreover, the FL of CDs is pH dependent (Figure S6, SI), suggesting that the as-prepared CDs is potential to be used as pH indicator.


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On the other hand, rare earth ions have low luminescent efficiency, but can emit strong FL owing to the antenna effect when specifically integrating with ligands.34 That occurred to us that Tb3+ modified the surface of CDs can not only keep its own coordination capacity, but preserve the intrinsic FL of CDs. Since there are rich amino and carboxyl groups on the as-prepared CDs, it is very easy to obtain CDs with surface integration of Tb3+ ions, which show controllable FL emissions with both intrinsic emissive features of CDs and the antenna effect of Tb3+ ions.30 The CDs-Tb are also characterized by XPS to determine their composition. The obtained results (Figure 1b, black curve) indicate that CDs-Tb are composed of carbon, nitrogen, oxygen and terbium atoms, confirming the successful modification of Tb3+ ions on the CDs. The as-prepared CDs-Tb show resembling excitation-independent FL properties to the original CDs (Figure S7, SI) and the maximum emission can be available at 459 nm with the maximum excitation at 350 nm which has a slight blue shift compared with original CDs. In addition, the introduction of Tb3+ ions can also enhance the FL intensity of CDs (Figure S8, SI). This phenomenon is because the bind of Tb3+ ions with the surface groups of CDs hampers the rotation of surface groups and enhances the πconjugation, which reduces the non-radiative decay.35-37 By calculating the radiative and non-radiative rate of CDs and CDs-Tb, we can clearly find that the radiative rate almost unchanged from 10.63 × 107 s-1 to 9.99 × 107 s-1, but the non-radiative rate obviously decreased from 3.38 × 107 s-1 to 0.50 × 107 s-1 (Table S1, SI) with the introduction of Tb3+ ions, thus the introduction of Tb3+ ions can significant improve the FL efficiency of CDs. The average FL lifetime (Figure 1e) of CDs is 7.14 ns within a typical FL decay level, while the CDs-Tb is obviously increased to 9.53 ns. The increased FL lifetime indicates the increase of transition time from excited state to ground state, which is owing to the significantly decreased non-radiative rate of CDs after the introduction of Tb3+ ions (Table S1, SI) The zeta potentials (Figure 1f) show that the CDs are considerably


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negatively charged, while the CDs-Tb display much positive potential due to the introduction of Tb3+ ions. Meanwhile, FT-IR spectrum (Figure 1c, red curve) of CDs-Tb shows slight change of the position of characteristic peaks, but the peak intensities of νC=O,

δN-H and νC-N have a significant difference with the original CDs, which are due to the change of dipole moments when the Tb3+ ions chelate with C=O and N-H bands. All the above results indicate the CDs-Tb have been successfully prepared. The fluorescent ratiometry of ppGpp FL titrations are conducted to evaluate the sensitivity of the CDs-Tb ratiometric FL chromophore for ppGpp. We firstly investigate the influence of pH on the ppGpp detection. Based on this result (Figure S9, SI), the most appropriate pH is 6.52. Figure 2a shows the FL response of the CDs-Tb when introducing different concentrations of

Figure 2 The sensitivity and selectivity of ppGpp detection. (a) The change of the FL spectra of CDs-Tb in the presence of ppGpp; (b)The sensitivity of ppGpp detection; (c) FL responses of the CDs-Tb in the absence and presence of 15 µM of ppGpp and 15 µM of phosphate radical and nucleotides; (d) FL responses of the CDs-Tb in the absence and presence of 15 µM of ppGpp and 100 µM of other ions. 1, Control; 2, Al3+; 3, Ba2+; 4, Ca2+; 5, Cr3+; 6, Cu2+; 7, K+; 8, Mg2+; 9, Na+; 10, Zn2+; 11, Cl-; 12, HCO3-; 13, I-; 14, SO32-; 15, SO42-; 16, Cys; 17, G-SH. cCDs-Tb, 0.05 mg/mL; pH, 6.52 (20 mM Tris-HCl); EX: 290 nm.


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ppGpp into aqueous solution. The emission spectrum of the CDs-Tb prior to ppGpp addition is dominated by the bands of CDs with the major emission peak at 459 nm, which is in agreement with that of the pure CDs solution. The FL lifetime (Figure S10, SI) of CDs-Tb is further increased in the presence of ppGpp, reaching 12.16 ns. With the increase of the ppGpp concentration, the intensity of the emission band at 459 nm remains unchanged, while the intensity of the emission bands of Tb3+ ions at 498 and 552 nm are greatly enhanced. The emission bands at 498 and 552 nm are attributed to the 5D4→7F6 and 5D4→7F5 transitions of Tb3+ ions, respectively.30 The most intense emission peak at 552 nm is sharp and sensitive to ppGpp, thus serving as the response signal for detecting ppGpp. The FL signal at 459 nm can act as a reference in the ratiometric chromophore to eliminate background interference and improve the detection sensitivity. Figure 2b shows that the FL intensity ratio (I552/I459) of the CDs-Tb is proportional to the concentration of ppGpp in the range of 0.5-15 µM. To our best knowledge, such a detection limit of 50 nM (3σ/K) is lower than the best performance of a FL chromophore for detecting ppGpp reported so far (Table S2, SI). Owing to the extreme structural similarity of ppGpp to normal nucleotides, selective detecting ppGpp is still in a difficult situation. Here, the problem is overcome, since the ppGpp-induced FL enhancement of the CDs-Tb is highly specific. As Figure 2c shows, 15 µM phosphate radical (PPi, Pi and PO43-) and another six nucleotides (ATP, CTP, UTP, GTP, GMP and GDP) are selected to study the FL performance of the CDs-Tb. Compared with the control group, FL intensity of CDs-Tb have no change when adding into other phosphate radical and nucleotides separately, demonstrating that the influence of phosphate radical and nucleotides is negligible. Similarly, phosphate radical and nucleotides have no influence on FL intensity of CDs-Tb in the presence of 15 µM ppGpp as well. The metal ions, anions and the reductants (G-SH and Cys) in biological samples


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are also used to investigate the selectivity of ppGpp detection (Figure 2d). All the interferents have no effect on FL intensity of CDs-Tb in the presence and absence of ppGpp. Except for these, we also investigate the effect of other chelating agents on the detection of ppGpp. The results (Figure S11, SI) show that some sulfydryl compounds (dimethyldithiocarbamate, diethyldithiocarbamate and thiourea) have no influence on the detection of ppGpp, while EDTA has a significant influence, which is resulting from the stronger binding ability between Tb3+ and EDTA than ppGpp. Therefore, the proposed fluorescent ratiometry has great potential to be used for detecting ppGpp in the complex samples based on the high selectivity. The selective binding mechanism of ppGpp with CDs-Tb The mechanism of specific sensing ppGpp with CDs-Tb as optical probes derives from the antenna effect of Tb3+ ions and specific recognition capacity of CDs. As shown in Figure 3a, ppGpp can efficiently enhance the emission intensity of Tb3+ ions. The phosphate groups originated from ppGpp can coordinate with rare earth ions.38 The ppGpp as the rare earth complex is easy to produce antenna effect with Tb3+ ions by energy transfer and significantly improve the FL of Tb3+ ions.30 Although the other analytes also have phosphate groups, the ppGpp molecule owns four phosphate groups, which have stronger coordinate capacity with Tb3+ ions on the surface of CDs compared with other nucleotides (Figure S12, SI). Meanwhile, the clamp shaped distribution of phosphate groups in the ppGpp may be easier to chelate with Tb3+ ions than line shaped distribution of phosphate groups in other nucleotides (such as GTP, GDP and GMP) (Figure S12, SI). Moreover, the ppGpp with lower electro-negativity is more easily to bind with positively charged CDs-Tb by electrostatic interaction (Figure S13-S14, SI) due to the more phosphate groups. Meanwhile, the FL of CDs (Figure 3b) has no change in the presence and absence of ppGpp. The above results indicate that the FL enhancement origins from the antenna effect between ppGpp and Tb3+ ions. In addition to ppGpp, the


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Figure 3 The mechanism of ppGpp detection. (a) FL responses of the Tb3+ ions in the absence and presence of ppGpp; (b) FL responses of the CDs in the absence and presence of ppGpp. cppGpp, 20 µM; EX: 290 nm. (c) FL intensities at 552 nm of Tb3+ ions in the presence of other nucleotides (20 µM). cTb3+, 1 mM; cppGpp, 20 µM; EX: 290 nm. (d) FL responses of the CDs-Tb in the presence of different nucleotides. cppGpp and cnucleotides, 15 µM; cCDs-Tb, 0.05 mg/mL; EX: 290 nm.

other nucleotides, especially GTP, can also enhance the FL of free Tb3+ ions (Figure 3c). Therefore, the free Tb3+ ions are improper to special detection of ppGpp. Interestingly, the CDs-Tb show the highly selectivity for ppGpp detection (Figure 3d), thus we believe that the highly selective recognition of ppGpp is resulting from the synergistic effect of antenna effect of Tb3+ ions and specific recognition capacity of CDs. Visual detection of ppGpp We also develop a facile and visual detection of ppGpp. For that purpose, the CDs-Tb solution is dropped into filter paper and the FL paper sensor is available when the filter paper is dried. Under the irradiation (254 nm) of a UV lamp, the entire paper presents the weak blue FL (Figure 4aA). With the increase of ppGpp concentration from 25 µM to 750 µM, the FL of CDs-Tb becomes more and more bright (Figure 4aB). In the meanwhile, we investigate the selectivity of ppGpp detection in the filter paper. Only ppGpp can


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Figure 4 CDs-Tb-based paper sensor for ppGpp visual detection. (a) Photographs (A) of the filter paper with the 10 µL CDs-Tb (1.0 mg/mL) upon the 254 nm UV lamp; photographs (B) of the filter paper staining with the CDs-Tb which contains 10 µL of ppGpp solution at different concentrations (from 1 to 8: 0, 25, 50, 75, 100, 250, 500 and 750 µM); photographs (C) of the filter paper staining with the CDs-Tb which contains 10 µL of 250 µM different nucleotides (from 1 to 8: control, ppGpp, ATP, CTP, UTP, GTP, GMP and GDP). (b) The 3D models of the images in (a).

efficiently enhance the FL of CDs-Tb when adding different nucleotides solution separately (Figure 4aC). Meanwhile, the three-dimensional (3D) models of the same field are used to obtain the quantitative FL intensity by Image J software, which clearly show that the FL of CDs-Tb can be significantly enhanced with the increase of ppGpp concentration and only ppGpp can improve the FL of CDs-Tb (Figure 4b). Therefore, the CDs-Tb-based paper sensor has a potential application for the visual detection of ppGpp due to its simplicity and rapidity. Moreover, the proposed method is applied in the detection of ppGpp in real samples by detecting recoveries after adding given amounts of ppGpp. The recoveries are varied from 101.9 % to 108.6 % in urine and from 98.5 to 110.8 % in serum, respectively (Table S3,


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SI), indicating the potential applications in the detection of ppGpp in the complex samples. Importantly, the proposed strategy owns the lowest limit of detection (LOD, 50 nM) than other methods, which can well meet the requirements of practical applications. CONCLUSIONS In summary, a rapid and convenient preparation route of CDs-Tb was developed in this work. Surface modification of Tb3+ ions on the CDs can not only keep its own coordination capacity, but preserve the intrinsic FL of CDs, enabling the CDs-Tb being a ratiometric FL chromophore.The as-prepared CDs-Tb can be used for detecting ppGpp, and the limit of detection reaches 50 nM which is the lowest value reached by FL chromophore reported so far. Notably, the CDs-Tb can specifically recognize ppGpp from its analogues as the result of the synergistic effect of antenna effect of Tb3+ ions and specific recognition capacity of CDs. Such a considerably high selectivity and sensitivity for detecting ppGpp enable the method possesses a broad application prospect in terms of detection of ppGpp. Furthermore, the rare earth ions-modified CDs have a great potential as a new type of ratiometric FL chromophore. ASSOCIATED CONTENT Supporting Information Characterization of the CDs and CDs-Tb, stability investigation and analysis details. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], [email protected]. Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENTS We are grateful for the financial support from the National Natural Science Foundation of China (NSFC, Grant No. 21535006). REFERENCES (1) Magnusson, L. U.; Farewell, A.; Nyström, T. Trends Microbiol. 2005, 13 (5), 236-242. (2) Hesketh, A.; Chen, W. J.; Ryding, J.; Chang, S.; Bibb, M. Genome Biol. 2007, 8 (8), 161179. (3) Braeken, K.; Moris, M.; Daniels, R.; Vanderleyden, J.; Michiels, J. Trends Microbiol. 2006, 14 (1), 45-54. (4) Fischer, M.; Zimmerman, T. P.; Short, S. A. Anal. Biochem. 1982, 121 (1), 135-139. (5) Takahashi, K.; Kasai, K.; Ochi, K. Proc. Natl. Acad. Sci. 2004, 101 (12), 4320-4324. (6) Jensen, K. F.; Houlberg, U.; Nygaard, P. Anal. Biochem. 1979, 98 (2), 254-263. (7) Rhee, H. W.; Lee, C. R.; Cho, S. H.; Song, M. R.; Cashel, M.; Choy, H. E.; Seok, Y. J.; Hong, J. I. J. Am. Chem. Soc. 2008, 130 (3), 784-785. (8) Zheng, L. L.; Huang, C. Z. Analyst 2014, 139 (23), 6284-6289. (9) Zhang, P.; Wang, Y.; Chang, Y.; Xiong, Z. H.; Huang, C. Z. Biosens. Bioelectron. 2013, 49, 433-437. (10) Liu, M. L.; Yang, L.; Li, R. S.; Chen, B. B.; Liu, H.; Huang, C. Z. Green Chem. 2017, 19 (15), 3611-3617. (11) Liu, M. L.; Chen, B. B.; Yang, T.; Wang, J.; Liu, X. D.; Huang, C. Z. Methods. Appl. Fluoresc. 2017, 5 (1), 015003. (12) Peng, J.; Gao, W.; Gupta, B. K.; Liu, Z.; Romero-Aburto, R.; Ge, L. H.; Song, L.; Alemany, L. B.; Zhan, X. B.; Gao, G. H.; Vithayathil, S. A.; Kaipparettu, B. A.; Marti, A. A.; Hayashi, T.; Zhu, J. J.; Ajayan, P. M. Nano Lett. 2012, 12 (2), 844-849. (13) Li, L. L.; Ji, J.; Fei, R.; Wang, C. Z.; Lu, Q.; Zhang, J. R.; Jiang, L. P.; Zhu, J. J. Adv. Funct. Mater. 2012, 22 (14), 2971-2979. (14) Essner, J. B.; Laber, C. H.; Ravula, S.; Polo-Parada, L.; Baker, G. A. Green Chem. 2016, 18 (1), 243-250. (15) Essner, J. B.; Laber, C. H.; Baker, G. A. J. Mater. Chem. A 2015, 3 (31), 16354-16360. (16) Lin, Z.; Xue, W.; Chen, H.; Lin, J. M. Anal. Chem. 2011, 83 (21), 8245-8251. (17) Huang, H. D.; Wei, H. J.; Zou, M. J.; Xu, X.; Xia, B.; Liu, F.; Li, N. Anal. Chem. 2015, 87 (5), 2748-2754. (18) Shi, B. F.; Su, Y. B.; Zhang, L. L.; Huang, M. J.; Li, X. F.; Zhao, S. L. Nanoscale 2016, 8 (20), 10814-10822. (19) Liu, Z. X.; Zhou, H. Y.; Wang, N.; Yang, T.; Peng, Z. W.; Wang, J.; Li, N.; Huang, C. Z. Sci. China Chem. 2017, DOI: 10.1007/s11426-017-9172-0.


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(20) Liu, H.; Yang, X. X.; Zheng, J. J.; Li, Y. F.; Huang, C. Z. Sci. China Chem. 2013, 43 (7), 895-900. (21) Lin, L. P.; Rong, M. C.; Lu, S. S.; Song, X. H.; Zhong, Y. X.; Yan, J. W.; Wang, Y. R.; Chen, X. Nanoscale 2015, 7 (5), 1872-1878. (22) Park, Y.; Yoo, J.; Lim, B.; Kwon, W.; Rhee, S. W. J. Mater. Chem. A 2016, 4 (30), 11582-11603. (23) Li, R. S.; Gao, P. F.; Zhang, H. Z.; Zheng, L. L.; Li, C. M.; Wang, J.; Li, Y. F.; Liu, F.; Li, N.; Huang, C. Z. Chem. Sci. 2017, 8, 6829-6835. (24) Wang, F.; Zheng, X.; Zhang, H.; Liu, C. Y.; Zhang, Y. G. Adv. Funct. Mater. 2011, 21 (21), 1027-1031. (25) Li, N.; Than, A.; Wang, X. W.; Xu, S. H.; Sun, L.; Duan, H. W.; Xu, C. J.; Chen, P. ACS Nano 2016, 10 (3), 3622-3629. (26) Yang, L.; Jiang, W. H.; Qiu, L. P.; Jiang, X. W.; Zuo, D. Y.; Wang, D. K.; Yang, L. Nanoscale 2015, 7 (14), 6104-6113. (27) Liu, C. J.; Zhang, P.; Zhai, X. Y.; Tian, F.; Li, W. C.; Yang, J. H.; Liu, Y.; Wang, H. B.; Wang, W.; Liu, W. G. Biomaterials 2012, 33 (13), 3604-3613. (28) Chen, B. B.; Liu, Z. X.; Zou, H. Y.; Huang, C. Z. Analyst 2016, 141 (9), 2676-2681. (29) Wu, W. T.; Zhan, L. Y.; Fan, W. Y.; Song, J. Z.; Li, X. M.; Li, Z. T.; Wang, R. Q.; Zhang, J. Q.; Zheng, J. T.; Wu, M. B. Angew. Chem. Int. Ed. 2015, 54 (22), 6540-6544. (30) Chen, H.; Xie, Y. J.; Kirillov, A. M.; Liu, L. L.; Yu, M. H.; Liu, W. S.; Tang, Y. Chem. Commun. 2015, 51 (24), 5036-5039. (31) Zhu, A. W.; Qu, Q.; Shao, X. L.; Kong, B.; Tian, Y. Angew. Chem. Int. Ed. 2012, 51 (29), 7185-7189. (32) Liu, S.; Tian, J. Q.; Wang, L.; Zhang, Y. W.; Qin, X. Y.; Luo, Y. L.; Asiri, A. M.; AlYoubi, A. O.; Sun, X. P. Adv. Mater. 2012, 24 (15), 2037-2041. (33) Jiang, K.; Sun, S.; Zhang, L.; Wang, Y. H.; Cai, C. Z.; Lin, H. W. ACS Appl. Mater. Interfaces 2015, 7 (41), 23231-23238. (34) Deoliveira, E.; Neri, C. R.; Serra, O. A.; Prado, A. G. S. Chem. Mater. 2007, 19 (22), 5437-5442. (35) Liu, Z. X.; Wu, Z. L.; Gao, M. X.; Liu, H.; Huang, C. Z. Chem. Commun. 2015, 52 (10), 2063-2066. (36) Chen, B. B.; Li, R. S.; Liu, M. L.; Zhang, H. Z.; Huang, C. Z. Chem. Commun. 2017, 53 (36), 4958-4961. (37) Lakowicz, J. R., Principles of fluorescence spectroscopy. Kluwer Academic/Plenum Publishers, New York, 2nd edn, 1999. (38) Zhao, H. X.; Liu, L. Q.; Liu, Z. D.; Wang, Y.; Zhao, X. J.; Huang, C. Z. Chem. Commun. 2011, 47 (9), 2604-2606.


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For TOC only


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Scheme 1. Illustration of the synthesis process of CDs-Tb for highly selective detection of ppGpp, in which CDs are first prepared from citric acid and triethylenetetramine (TETA) by the hydrothermal method. 814x598mm (150 x 150 DPI)


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Figure 1 The morphology, elemental analysis, FL properties and zeta potentials of the CDs. (a) TEM image of CDs (Inset: particle size distribution ); (b) XPS spectra of CDs and CDs-Tb; (c) FT-IR spectra of CDs and CDs-Tb; (d) absorption and FL spectra of CDs (Inset: photograph of CDs solution under the excitation of the 365 nm UV light lamp). (e) FL lifetimes of CDs and CDs-Tb; (f) zeta potentials of CDs and CDs-Tb. 281x165mm (150 x 150 DPI)



Figure 2 The sensitivity and selectivity of ppGpp detection. (a) The change of the FL spectra of CDs-Tb in the presence of ppGpp; (b)The sensitivity of ppGpp detection; (c) FL responses of the CDs-Tb in the absence and presence of 15 µM of ppGpp and 15 µM of phosphate radical and nucleotides; (d) FL responses of the CDs-Tb in the absence and presence of 15 µM of ppGpp and 100 µM of other ions. 1, Control; 2, Al3+; 3, Ba2+; 4, Ca2+; 5, Cr3+; 6, Cu2+; 7, K+; 8, Mg2+; 9, Na+; 10, Zn2+; 11, Cl-; 12, HCO3-; 13, I-; 14, SO32-; 15, SO42-; 16, Cys; 17, G-SH. cCDs-Tb, 0.05 mg/mL; pH, 6.52 (20 mM Tris-HCl); EX: 290 nm. 213x177mm (150 x 150 DPI)


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Figure 3 The mechanism of ppGpp detection. (a) FL responses of the Tb3+ ions in the absence and presence of ppGpp; (b) FL responses of the CDs in the absence and presence of ppGpp. cppGpp, 20 µM; EX: 290 nm. (c) FL intensities at 552 nm of Tb3+ ions in the presence of other nucleotides (20 µM). cTb3+, 1 mM; cppGpp, 20 µM; EX: 290 nm. (d) FL responses of the CDs-Tb in the presence of different nucleotides. cppGpp and cnucleotides, 15 µM; cCDs-Tb, 0.05 mg/mL; EX: 290 nm. 224x187mm (145 x 145 DPI)



Figure 4 CDs-Tb-based paper sensor for ppGpp visual detection. (a) Photographs (A) of the filter paper with the 10 µL CDs-Tb (1.0 mg/mL) upon the 254 nm UV lamp; photographs (B) of the filter paper staining with the CDs-Tb which contains 10 µL of ppGpp solution at different concentrations (from 1 to 8: 0, 25, 50, 75, 100, 250, 500 and 750 µM); photographs (C) of the filter paper staining with the CDs-Tb which contains 10 µL of 250 µM different nucleotides (from 1 to 8: control, ppGpp, ATP, CTP, UTP, GTP, GMP and GDP). (b) The 3D models of the images in (a). 153x166mm (150 x 150 DPI)


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Terbium (III) modified fluorescent carbon dots for highly selective and

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