Non-Redox Modulated Fluorescence Strategy for Sensitive and

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Analytical Chemistry

A Non-Redox Modulated Fluorescence Strategy for Sensitive and Selective Ascorbic Acid Detection with Highly Photoluminescent Nitrogen-Doped Carbon Nanoparticles via Solid-State Synthesis Xiaohua Zhuab, Tingbi Zhaoa, Zhou Nieb*, Yang Liua*, Shouzhuo Yaob a

Department of Chemistry, Beijing Key Laboratory for Analytical Methods and

Instrumentation, Tsinghua University, Beijing 100084, China b

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of

Chemistry and Chemical Engineering, Hunan University, Changsha 410082, People’s Republic of China

*to whom corresponding should be addressed. Tel: 86-10-62798187; Fax: 86-10-62771149 Email: [email protected], [email protected]

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Abstract: Highly photoluminescent nitrogen-doped carbon nanoparticles (N-CNPs) were prepared by a simple and green route employing sodium alginate as carbon source and tryptophan as both of nitrogen source and functional monomer. The as-synthesized N-CNPs exhibited excellent water solubility and biocompatibility with a fluorescence quantum yields of 47.9 %. The fluorescence of the N-CNPs was intensively suppressed by the addition of ascorbic acid (AA). The mechanism of the fluorescence suppression of the N-CNPs was investigated, and the synergistic action of inner filter effect (IFE) and static quenching effect (SQE) contributed to the intensive fluorescence suppression, which was different from those reported for the traditional redox based fluorescent probes. Owing to the spatial effect and hydrogen bond between the AA and the groups on the N-CNP surface, excellent sensitivity and selectivity for AA detecting was obtained in a wide linear relationship from 0.2 μM to 150 μM. The detection limit was as low as 50 nM (signal-to-noise ratio of 3). The proposed sensing systems also represented excellent sensitivity and selectivity for AA analysis in human biological fluids, providing a valuable platform for AA sensing in clinic diagnostic and drug screening. Keywords: Carbon nanoparticles; Non-redox fluorescence probe; Ascorbic acid; Inner filter effect

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Introduction Ascorbic acid (AA) is an important micronutrient that is essential for humans and animals, and has been widely used in various areas including chemical, biological, pharmaceutical and nutritional systems. AA is an antioxidant, free-radical scavenger, and one of the most important neurochemicals in cerebral systems,1-3 and is also a medication for scurvy, drug poisoning, liver disease, allergic reactions and atherosclerosis, that helps to promote healthy cell development and normal tissue growth.4,5 The lack of AA will result in scurvy, and excessive intake of AA can lead to urinary stone, diarrhea, and stomach convulsion.6,7 As a result, the rapid, sensitive, and selective detection of AA level is of significance in cases of medical assay and diagnosis. Up to now, several methods have been developed for the determination of AA, including electrochemistry,8-10 chromatography,11-13 capillary electrophoresis,14,15 colorimetry,16,17 and fluorescence spectroscopy.18,19 Among them, the fluorescence method possesses the advantages, such as high sensitivity, fast analysis, and good reproducibility, and receives extensive attention. Fluorescence determination of AA has been achieved with the help of different indicators, such as enzyme20 and organic dye.21,22 In the past decade, due to their unique, novel properties and biomedical applications, nanomaterials have gained increasing interest.23,24 Some novel and efficient fluorescence nanoprobes based on the specific reaction between the nanomaterials and AA provide a promising way for the determination of AA.25-28 For instance, Yan’s group developed a CdTe QDs fluorescent probe for the selective 3



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detection of AA after quenching by a certain amount of KMnO4.29 Tang’s group designed

cobalt

oxyhydroxide

(CoOOH)-modified

persistent

luminescence

nanoparticles (Sr2MgSi2O7:1% Eu, 2% Dy) for determination and screening of AA in living cells and in vivo based on the specific reaction of CoOOH.30 Mao’s group investigate the mechanism of single-layer MnO2 nanosheets suppressing fluorescent of 7-hydroxycoumarin and demonstrate a new fluorescent method for in vivo sensing of AA in rat brain.31 Very recently, Li and coworkers32 report a novel and efficient fluorescent probe based on carbon dots modified hexagonal CoOOH nanoflakes for monitoring of cerebral AA in brain microdialysate. In these strategies, most of fluorescent nanoprobes were served as the oxidizer to oxidize AA, and the redox reaction of nanoprobes leads to the intensive fluorescence changes. In this way, some reductive species such as glutathione and cysteine can also disturb the analysis of AA in biological fluids. In addition, the complicated and harsh fabrication procedures and biocompatibility of the nanoprobes also limited their applications in clinic detection and diagnostic.31 Consequently, it is still in high demand to design simple, efficient, and biocompatible fluorescent probes for AA analysis in physiological and pathological processes. Recently, fluorescent carbon nanoparticles (CNPs) have attracted tremendous attention because of their special advantages, such as low toxicity, high fluorescent activity, robust chemical inertness, and excellent photostability.33-36 CNPs are more superior to traditional organic dyes and semiconductor quantum dots in terms of aqueous solubility, biocompatibility and easy functionality, which make them 4


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fascinating in biosensing and imaging applications.37-39 For example, Tian’s group employing N-(2-aminoethyl)-N,N,N-tris (pyridine-2-ylmethyl) ethane-1,2-diamine chemically functionalized on the surface of CNPs to enhance the selectivity, and applying the CNPs-based probe to image and sense Cu2+ in living cells.40 Shi et al developed CNPs-based tunable ratiometric fluorescent pH sensor for the intracellular pH pattern of HeLa cells.41 As a result, a numerous efforts have been focused on the preparation of CNPs as well as their bioimaging applications. Though great progresses have been made in these years, some distinct limitations of currently available CNPs still exist, such as the relatively low yield (most of the reported CNPs are milligram level), low fluorescent quantum yields (generally less than 10%), and broad full width at half maximum of the fluorescent peak (mostly larger than 100 nm). It is still a challenge to the synthesis of highly fluorescent water-soluble CNPs with a simple and effective method. Herein, a facile solid-phase synthesis strategy for fabrication of highly photoluminescent nitrogen-doped carbon nanoparticles (N-CNPs) was developed using sodium alginate as the carbon source and tryptophan as both of nitrogen source and functional monomer (Scheme 1A). The as-synthesized N-CNPs exhibited excellent biocompatibility with a fluorescence quantum yields of 47.9 %. The fluorescence of the as-synthesized N-CNPs can be intensively suppressed by AA (Scheme 1B), attributing to the synergistic action of inner filter effect (IFE) and static quenching effect (SQE). Because of the spatial effect and hydrogen bond between the AA and the groups on the N-CNP surface, excellent sensitivity and selectivity for AA 5



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detecting were obtained in a wide linear relationship from 0.2 μM to 150 μM with a detection limit as low as 50 nM. The determination AA using the N-CNPs as nanoprobes in human blood and urine samples were also studied, showing a great promising in clinic diagnostic and cellular imaging applications.

EXPERIMENTAL SECTION Chemicals Sodium alginate, AA, dopamine (DA), uric acid (UA), adenosine 5’-triphosphate (ATP), L-glutathione (GSH), bovine serum albumin (BSA), hemoglobin (He), glucose, catechol, hydroquinone, nicotinamide adenine dinucleotide (NADH), quinine sulfate and all the amino acid were purchased from Aladdin Chemistry Co. Ltd. (Shanghai, China). D-isoascorbic acid and dehydroascorbic acid were purchased from Sigma and used as supplied. KCl, NaCl, Ca(NO3)2, MgSO4, NH4F, and NaNO2 were bought from Beijing Chemical Reagent Co. Ltd. (Beijing, China). All other reagents were of analytical grade and used as received without further purification. All solutions were prepared with double distilled water. Apparatus and Characterization The morphologies and sizes of N-CNPs were characterized by transmission electron microscopy (TEM, Hitachi-600, Japan). Fourier transform infrared spectra (FT-IR) were recorded on a Fourier transform spectrometer (TENSOR27, Bruker, Germany). X-ray photoelectron spectroscopy (XPS) was performed on an ESCALAB 250 photoelectron spectrometer (Thermo Fisher Scientific, USA) with Al Kα (1486.6 6


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eV) as the X-ray source. All fluorescence spectra were surveyed on an RF-5301PC fluorescence spectrophotometer (Shimadzu, Kyoto, Japan). The UV-vis spectra were obtained by a Hitachi U-3900 UV-vis spectrophotometer (Japan). Fluorescence decay curves were performed with the time-correlated single photo counting technique on the combined steady state and lifetime spectrometer (Edinburgh Instruments FLSP920). Preparation of N-CNPs In a typical synthesis, 2.0 g of sodium alginate and 1.0 g of tryptophan were mixed in an agate mortar and ground to a uniform powder. Then the mixture was transferred into a 25 mL Teplon lined autoclave and heated at 220 °C for 6 h. The resultant brown mixture (yield ca. 66 %) was dissolved with ethanol. The brownish-yellow supernatant was collected by removing the large dots through centrifugation at 10 000 rpm for 10 min, and mixed with methylbenzene (ethanol/methylbenzene volume ratio was 1:3) and centrifuged at 12 000 rpm for 30 min. The precipitate was dried at 60 oC and light brownish powder of N-CNPs was obtained. Detection of AA N-CNPs solution (10 μg mL-1) was diluted by the phosphate buffered solution (PBS, 50 mM, pH 6.0). For the detection of AA, different concentrations of AA were added to the N-CNPs solution at room temperature. For the selectivity of AA detection, 5 mM for Na+, K+, Ca2+, NH4+, Mg2+,Cl-, F-, NO2-, SO42- and glucose, 50 μM for hydroquinone, GSH, and all the amino acid, 30 μM DA and UA, 5 μM for 7



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ATP and NADH, 50 μg mL-1 for BSA and Hb were added to each of the above N-CNPs solutions, respectively. After that the fluorescence spectra of the mixture were recorded (excited at 270 nm).

RESULTS AND DISCUSSION Characterization of N-CNPs The morphologies of the as-synthesized CNPs was characterized by TEM. As shown in Figure 1A, the CNPs is nearly monodispersed with spherical morphology. The average size of CNPs is 13.3 nm (Figure 1A insert). Figure 1B shows the FT-IR spectra of the CNPs. The peak at 3400 cm-1 is assigned to stretching vibrations of O-H, and the peak of the C-O group conjugated with condensed aromatic carbons at 1095 cm-1 appear. The peak at 3250 cm-1 corresponds to the N-H vibrations.42 These results indicate that there are abundant of hydroxyl and amino groups on the surface of the as-synthesized CNPs, which made the CNPs hydrophilic. In addition, two peaks at 1250 cm-1 and 1310 cm-1 are observed, which are ascribed to C-N stretching vibrations and the aromatic C=N heterocycles stretching vibrations, respectively.43 Since there is no C=N group in the precursor molecules, the C=N group may come from the doped nitrogen atom in the form of aromatic heterocycles during carbonization process. The fact suggests that the CNP could be chemically doped with nitrogen atom. The as-synthesized CNPs were further characterized by XPS. Three strong peaks at 532.0 eV, 401.1 eV, and 286.1 eV are observed in the XPS spectra (Figure S1 A, Supporting Information), which are attributed to O1s, N1s, and C1s, 8


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respectively. The C 1s spectrum (Figure S1 B, Supporting Information) shows three fitted peaks at 284.4, 285.5, and 288.5 eV, which are attributed to C-C/C=C, C-N/C-O, and C=O groups, respectively.44,45 The two fitted peaks at 530.8 and 532.0 eV in O 1s spectrum (Figure S1 C, Supporting Information) are attributed to C=O and C-OH groups, respectively.43,46 The XPS spectrum of N1s (Figure 1C) exhibits two fitted peaks at 398.5 and 399.5 eV, which are associated with nitrogen in a pyridine-like, pyrrolic-like nitrogen, respectively.46-48 The N/C atomic ratio was calculated to be 4.05 %. The facts demonstrated that the nitrogen atoms were doped as the form of pyridine-like nitrogen in the CNPs. The N-CNP was also analyzed by Raman spectra. A peak observed at 1540 cm-1 in the Raman spectra is related to the E2g mode of graphite (G band) from the vibration of sp2-bonded carbon. In addition, the peak at 1320 cm-1 is assigned to the D band from the sp3 defects in carbogenic material (Figure S2, Supporting Information). Compared with that of CNPs without doping, a blue shift on the peaks of the D and G band of the N-CNPs is observed, suggesting that the nitrogen atoms is intercalated the conjugated carbon backbone of the CNPs.49 Optical Properties of the N-CNPs The UV-Vis spectra and fluorescence spectra of the N-CNPs were also studied. An absorption peak at ca. 270 nm is observed in the UV-Vis spectra of the N-CNPs (Figure 2 A, curve a), which is assigned to the π-π* transition of aromatic sp2 domains. In addition, there is an absorption peak at around 365 nm, corresponding to the transition of n-π* transition of the C=O and C=N bond.18,44 Curve b in Figure 2A shows the fluorescence emission spectrum of the N-CNP. There is a strong peak at 9



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440 nm with an excitation wavelength of 270 nm. The quantum yields of the N-CNP is determined to be 47.9 % using quinine sulfate as a reference and the full width at half maximum of the fluorescence emission spectrum is 68 nm. It is known that doping and/or surface passivation are effective methods to change the electronic density of carbon nanoparticles and enhance the quantum yields.50,51 In order to investigate the influence of the quantity of doped nitrogen on quantum yields, the CNPs were synthesized with various molar ratios sodium alginate and tryptophan as precursor. The highest quantum yields of N-CNPs was obtained with the molar ratio of 2:1 (Figure S3, Supporting Information), which was significantly higher than that of CNPs without doping. The as-prepared N-CNP in this method presented excellent stability. The fluorescence intensity of the N-CNP has no change after 2 months storage or even under the light illumination for 1 hour (Figure S4 in Supporting Information). In addition, the fluorescence intensity keep stable at the concentrations of NaCl as high as 1 M, showing the excellent photostability and salt-tolerance of the N-CNPs (Figure S5 in Supporting Information). Moreover, the cytotoxicity of N-CNPs was studied by determining the cell viability of Hela cells in vitro. The methylthiazol tetrazolium (MTT) assay results indicate that the viability of cells remained unchanged when the cells are exposed to N-CNPs in the range of the cytotoxicity of 2.0 - 500 μg mL-1 (Figure S6, Supporting Information). The result suggests that the N-CNPs has excellent biocompatible, which may be resulted from the plentiful hydrophilic groups on the surface of the as-synthesized N-CNPs. 10


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Fluorescence response of the N-CNPs to the AA Figure 2A shows the UV-Vis spectra and the fluorescence emission spectra of N-CNP in the presence of AA. It can be seen that the fluorescence intensity of the N-CNPs decreased to about 80 % of the original fluorescence intensity after the addition of 100 μM AA, indicating that AA can effectively suppress the fluorescence of N-CNPs (Figure 2A, curve c). A new UV-Vis spectrum peak at ca. 265 nm is observed when the AA is added into the N-CNPs solution (Figure 2A, curve d). The observed UV-Vis spectrum peak overlap with that of N-CNPs, and the absorbance intensity is increased with the increases of AA concentrations (Figure S7, Supporting Information). The fact suggests that the AA induced fluorescence suppression of N-CNPs may be obedient to the IFE mechanism. On the basis of the cuvette geometry used in the fluorescent measurements (Figure 2B insert) and of the absorption characteristics of the mixture of N-CNPs and AA, the IFE is estimated with equation 1.31,52

Fcor 2.3dAex 2.3sAem gA  10 em dAex Fobsd 1  10 sAem 1  10

(1)

Where, Fobsd is the measured maximum fluorescence intensity and Fcor is the corrected maximum fluorescence intensity by removing IFE from Fobsd; Aex and Aem represent the absorbance at the excitation wavelength (λex = 270 nm) and maximum emission wavelength (λem = 440 nm), respectively; s is the thickness of excitation beam (0.10 cm), g is the distance between the edge of the excitation beam and the edge of the cuvette (0.40 cm in this case), and d is the width of the cuvette (1.00 cm). The maximum value of the correction factor could not exceed 3; otherwise the 11



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correction is not convincing. The correction factor of IFE at each concentration of AA was calculated (Table S1, Supporting Information). After the IFE was removed from the totally observed suppressed fluorescence, the suppressed efficiency for the totally observed and the corrected (i.e., after removing IFE) fluorescence of AA was figured out, as demonstrated in Figure 2B. The results demonstrate that approximately 80 % of the suppressed effect came from the IFE of AA. After removing the IFE, the remaining suppressed effect may be come from the quenching effect of AA toward N-CNPs. To further reveal the mechanism of fluorescence quenching, time-resolved fluorescence spectra of the N-CNPs in the absence and presence of AA were measured. As shown in Figure 2C, the fluorescence lifetime of the N-CNPs remained constant with the addition of AA, which implied that the fluorescence quenching of N-CNPs by AA may also obey static quenching mechanism.53-55 Thus, both IFE and SQE mechanism may be responsible for the fluorescence suppression of N-CNPs caused by AA. The results suggest that the N-CNPs can be used as a sensitive fluorescent probe for AA detection. Optimizing experimental conditions of the N-CNPs to the AA The response rate of the fluorescence signal of the N-CNPs to AA was first monitored. As shown in Figure S8 (Supporting Information), the fluorescence intensity of the N-CNPs rapidly decreased as soon as the AA was added into the N-CNPs and kept stable during the following 30 min, implying a promising application in a fast sensing of AA without strict time control. Besides, the pH value did not only affect the fluorescent intensity of the original N-CNPs solution, but also 12


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the subsequent fluorescence quenching by AA. The effects of the N-CNPs with AA reaction pH values was also optimized, and the results (Figure S9, Supporting Information) reveal that the value of F/F0 in PBS with pH 6.0 is higher than those of other pH values, so pH 6.0 was chosen for subsequent study. Detection AA with N-CNPs. To ensure the presented sensing system can be used for sensitive quantiﬁcation of AA, the ﬂuorescence responses induced by AA at different concentrations were evaluated. Under the optimal experimental conditions, the linear response range and the detection limit of the sensing system were measured. As shown in Figure 3A, when the concentration of AA increased, the fluorescence intensity of N-CNPs is decreased. There is a good logarithmic correlation between the quenching efficiency (F0/F) and the concentration of AA in the range from 0.2 to 150 μM (Figure 3B, insert) with a correlation equation 2:

logF0 / F   0.0065c M   0.0307 , R  0.998

(2)

where F0 and F are fluorescence intensities at 440 nm in the absence and presence of AA, respectively. The detection of limit (signal-to-noise ratio of S/N = 3) is estimated to be 50 nM. The comparison of the analytical performance of AA determination at the developed fluorescence probe with other methods reported previously is summarized in Table S2. It can be seen that the present N-CNPs nanoprobe exhibited wider linear range and lower detection limit, which may be attribute to the synergism of IFE and SQE, which shows great promising for AA detection in clinic diagnostics. 13



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Selectivity of N-CNPs for AA detection The selectivity of this biosensor for AA was evaluated in the present of several interferences. As shown in Figure 4, the fluorescence intensity of the N-CNPs exhibited significant decrease by adding AA to the solution, whereas other ions (including Na+, K+, Ca2+, NH4+, Mg2+,Cl-, F-, NO2-, and SO42-), small biomolecules (such as glucose, DA, UA, ATP, NADH, and hydroquinone), peptide (GSH), protein (BSA and Hb), and most of amino acids do not cause obvious fluorescence changes. It is noted that the reductive species such as GSH and cysteine have no obvious influences on the determination of AA, suggesting the detection mechanism of AA using this nanoprobe is different from the traditional redox based fluorescent probes. To further demonstrate the selectively toward AA in this system, the interaction of three AA analogues D-isoascorbic acid, dehydroascorbic acid, and catechol with N-CNPs were also investigated (Figure 4C). It is found that D-isoascorbic acid caused comparably fluorescence quenching efficiency, and catechol also caused slightly quenching the fluorescence intensity of N-CNPs. In contrast, dehydroascorbic acid caused negligible fluorescence quenching efficiency of N-CNPs. These results indicate that the ring structure and the enediol group of AA play an important role here. As a result, the excellent selectivity of the biosensor toward AA detection may be owing to the spatial effect and hydrogen band interaction between the surface of N-CNPs and AA. The excellent specificity combined with high sensitivity and fast response of the N-CNPs to AA suggests that this method might be directly applied to detecting AA in real samples. 14


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Application of the proposed sensing system in biological assay Combined with the good properties of excellent selectivity, high sensitivity, and low detection limit of the proposed sensing system, the application of the biosensor for AA detection in biological fluids was evaluated. The biological samples were spiked with standard solutions containing different concentrations of AA, and the fluorescence spectra was record. It is observed that the fluorescence intensity of the N-CNPs are decreased with increased concentration of AA from 5 μM to 40 μM, and well linear relationships are obtained by plotting the logarithm values of (F0/F) as a function of the concentrations of AA (Figure 5), where F0 and F are the fluorescence intensities of N-CNPs in the absence and presence of AA, respectively. In spite of the interference from numerous ions and organics existing in biological fluids, this sensing system can distinguish between biological fluids and that spiked with 5 μM AA, satisfying the practical AA detection in real samples. These results imply that the AA probe is likely to be capable of practically useful AA detection upon further development.

Conclusions In summary, highly photoluminescent nitrogen-doped carbon nanoparticles (N-CNPs) were synthesized by a facile and green solid-phase synthesis method. The N-CNPs were water-soluble and remarkably stable against extreme ionic strengths and light illumination, and could serve as a novel fluorescence probe for AA sensing. The mechanism of the fluorescence suppression of the N-CNPs was investigated, and 15



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the synergistic action of inner filter effect (IFE) and static quenching effect (SQE) were contributed to the intensive fluorescence suppression, which was relatively different from those reported for the traditional redox based fluorescent probes. This sensing system also possesses high sensitivity, high selectivity, rapid detection, and wide linear response range, and has been successfully used for the analysis of the biological fluids samples.

Acknowledgements This work was financially supported by the National Natural Science Foundation of China (NO. 21375073, NO. 21235004) and National Basic Research Program of China (NO. 2011CB935704, 2013CB934004), and Tsinghua University Initiative Scientific Research Program (2014z21027). Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org.

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References (1) Bohndiek, S. E.; Kettunen, M. I.; Hu, D. E.; Kennedy, B. W.; Boren, J.; Gallagher, F. A.; Brindle, K. M. J. Am. Chem. Soc. 2011, 133, 11795-11801. (2) Yin, R.; Mao, S. Q.; Zhao, B.; Chong, Z.; Yang, Y.; Zhao, C.; Zhang, D.; Huang, H.; Gao, J.; Li, Z.; Jiao, Y.; Li, C.; Liu, S.; Wu, D.; Gu, W.; Yang, Y. G.; Xu, G. L.; Wang, H. J. Am. Chem. Soc. 2013, 135, 10396-10403. (3) Eggersdorfer, M.; Laudert, D.; Letinois, U.; McClymont, T.; Medlock, J.; Netscher, T.; Bonrath, W. Angew. Chem., Int. Ed. 2012, 51, 12960-12990. (4) Heller, R.; Munscher-Paulig, F.; Grabner, R.; Till, U. J. Biol. Chem. 1999, 274, 8254-8260. (5) Kim, W. S.; Dahlgren, R. L.; Moroz, L. L.; Sweedler, J. V. Anal. Chem. 2002, 74, 5614-5620. (6) Massey, L. K.; Liebman, M.; Kynast-Gales, S. A. J. Nutr. 2005, 135, 1673-1677. (7) Zhao, Y.; Li, Y.; Wang, Y.; Zheng, J.; Yang, R. RSC Adv. 2014, 4, 35112-35115. (8) Gao, X.; Yu, P.; Wang, Y.; Ohsaka, T.; Ye, J.; Mao, L. Anal. Chem. 2013, 85, 7599-7605. (9) Bali Prasad, B.; Jauhari, D.; Tiwari, M. P. Biosens. Bioelectron. 2013, 50, 19-27. (10) Fernandes, D. M.; Teixeira, A.; Freire, C. Langmuir 2015, 31, 1855-1865. (11) Kishida, E.; Nishimoto, Y.; Kojo, S. Anal. Chem. 1992, 64, 1505-1507. (12) Lykkesfeldt, J. Anal. Biochem. 2000, 282, 89-93. (13) Frenich, A. G.; Torres, M. E. H.; Vega, A. B.; Vidal, J. L. M.; Bolanos, P. P. J. Agric. Food Chem. 2005, 53, 7371-7376. 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(14) Peng, Y.; Zhang, Y.; Ye, J. J. Agric. Food Chem. 2008, 56, 1838-1844. (15) Dong, S.; Zhang, S.; Cheng, X.; He, P.; Wang, Q.; Fang, Y. J. Chromatogr. A 2007, 1161, 327-333. (16) Wang, G.; Chen, Z.; Chen, L. Nanoscale 2011, 3, 1756-1759. (17) Li, L.; Huang, J.; Wang, T.; Zhang, H.; Liu, Y.; Li, J. Biosens. Bioelectron. 2010, 25, 2436-2441. (18) Zheng, M.; Xie, Z.; Qu, D.; Li, D.; Du, P.; Jing, X.; Sun, Z. ACS Appl. Mater. Interfaces 2013, 5, 13242-13247. (19) Rong, M.; Lin, L.; Song, X.; Wang, Y.; Zhong, Y.; Yan, J.; Feng, Y.; Zeng, X.; Chen, X. Biosens. Bioelectron. 2015, 68, 210-217. (20) Malashikhina, N.; Pavlov, V. Biosens. Bioelectron. 2012, 33, 241-246. (21) Ishii, K.; Kubo, K.; Sakurada, T.; Komori, K.; Sakai, Y. Chem. Commun. 2011, 47, 4932-4934. (22) Maki, T.; Soh, N.; Nakano, K.; Imato, T. Talanta 2011, 85, 1730-1733. (23) Li, K.; Wang, K.; Qin, W.; Deng, S.; Li, D.; Shi, J.; Huang, Q.; Fan, C. J. Am. Chem. Soc. 2015, 137, 4292-4295. (24) Xu, Y.; Li, K.; Qin, W.; Zhu, B.; Zhou, Z.; Shi, J.; Wang, K.; Hu, J.; Fan, C.; Li, D. Anal. Chem. 2015, 87, 1968-1973. (25) Di, W.; Shirahata, N.; Zeng, H.; Sakka, Y. Nanotechnology 2010, 21, 365501. (26) Liu, S.; Hu, J.; Su, X. Analyst 2012, 137, 4598-4604. (27) Wang, X.; Wu, P.; Hou, X.; Lv, Y. Analyst 2013, 138, 229-233. (28) Liu, C.-P.; Wu, T.-H.; Liu, C.-Y.; Cheng, H.-J.; Lin, S.-Y. J. Mater. Chem. B 2015, 18


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3, 191-197. (29) Chen, Y. J.; Yan, X. P. Small 2009, 5, 2012-2018. (30) Li, N.; Li, Y.; Han, Y.; Pan, W.; Zhang, T.; Tang, B. Anal. Chem. 2014, 86, 3924-3930. (31) Zhai, W.; Wang, C.; Yu, P.; Wang, Y.; Mao, L. Anal. Chem. 2014, 86, 12206-12213. (32) Li, L.; Wang, C.; Liu, K.; Wang, Y.; Liu, K.; Lin, Y. Anal. Chem. 2015, 87, 3404-3411. (33) Zhu, S.; Meng, Q.; Wang, L.; Zhang, J.; Song, Y.; Jin, H.; Zhang, K.; Sun, H.; Wang, H.; Yang, B. Angew. Chem., Int. Ed. 2013, 52, 3953-3957. (34) Bourlinos, A. B.; Zbořil, R.; Petr, J.; Bakandritsos, A.; Krysmann, M.; Giannelis, E. P. Chem. Mater. 2012, 24, 6-8. (35) Ding, C.; Zhu, A.; Tian, Y. Acc. Chem. Res. 2014, 47, 20-30. (36) Qian, Z.; Ma, J.; Shan, X.; Feng, H.; Shao, L.; Chen, J. Chem. Eur. J. 2014, 20, 2254-2263. (37) Zhang, P.; Xue, Z.; Luo, D.; Yu, W.; Guo, Z.; Wang, T. Anal. Chem. 2014, 86, 5620-5623. (38) Wang, L.; Zhou, H. S. Anal. Chem. 2014, 86, 8902-8905. (39) Bao, L.; Liu, C.; Zhang, Z. L.; Pang, D. W. Adv. Mater. 2015, 27, 1663-1667. (40) Zhu, A.; Qu, Q.; Shao, X.; Kong, B.; Tian, Y. Angew. Chem., Int. Ed. 2012, 51, 7185-7189. (41) Shi, W.; Li, X.; Ma, H. Angew. Chem., Int. Ed. 2012, 51, 6432-6435. 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(42) Zhu, X.; Zuo, X.; Hu, R.; Xiao, X.; Liang, Y.; Nan, J. Mater. Chem. Phys. 2014, 147, 963-967. (43) Wang, Z. X.; Ding, S. N. Anal. Chem. 2014, 86, 7436-7445. (44) Dong, Y.; Pang, H.; Yang, H. B.; Guo, C.; Shao, J.; Chi, Y.; Li, C. M.; Yu, T. Angew. Chem., Int. Ed. 2013, 52, 7800-7804. (45) Zhu, X.; Xiao, X.; Zuo, X.; Liang, Y.; Nan, J. Part. Part. Syst. Charact. 2014, 31, 801-809. (46) Zhang, H.; Chen, Y.; Liang, M.; Xu, L.; Qi, S.; Chen, H.; Chen, X. Anal. Chem. 2014, 86, 9846-9852. (47) Li, Y.; Zhao, Y.; Cheng, H.; Hu, Y.; Shi, G.; Dai, L.; Qu, L. J. Am. Chem. Soc. 2012, 134, 15-18. (48) Barman, S.; Sadhukhan, M. J. Mater. Chem. 2012, 22, 21832-21837. (49) Hu, C.; Liu, Y.; Yang, Y.; Cui, J.; Huang, Z.; Wang, Y.; Yang, L.; Wang, H.; Xiao, Y.; Rong, J. J. Mater. Chem. B 2013, 1, 39-42. (50) Xu, Y.; Wu, M.; Liu, Y.; Feng, X. Z.; Yin, X. B.; He, X. W.; Zhang, Y. K. Chem. Eur. J. 2013, 19, 2276-2283. (51) Ayala, P.; Arenal, R.; Loiseau, A.; Rubio, A.; Pichler, T. Rev. Mod. Phys. 2010, 82, 1843-1885. (52) Gauthier, T. D.; Shane, E. C.; Guerin, W. F.; Seitz, W. R.; Grant, C. L. Environ. Sci. Technol. 1986, 20, 1162-1166. (53) Bai, J. M.; Zhang, L.; Liang, R. P.; Qiu, J. D. Chem. Eur. J. 2013, 19, 3822-3826. (54) Shen, P.; Xia, Y. Anal. Chem. 2014, 86, 5323-5329. 20


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(55) Wu, W.; Zhou, T.; Berliner, A.; Banerjee, P.; Zhou, S. Angew. Chem., Int. Ed. 2010, 49, 6554-6558.

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FIGURE CAPTIONS Scheme 1 (A) The preparation procedures of N-CNPs using sodium alginate and tryptophan as precursors. (B) Working principle for AA sensing. Figure 1 (A) TEM image of the N-CNPs. The insert is the corresponding size distribution histograms. (B) FT-IR spectra and (C) XPS N 1s spectra of the as synthesized N-CNPs Figure 2 (A) UV-Vis spectra (a, d) and fluorescence spectra (b, c) of 10 μg mL-1 N-CNPs solution in the absence (a, b) and presence (c, d) of 100 μM AA. (B) Suppressed efficiency (F/F0) of observed (black curve) and corrected (red curve) measurements for N-CNPs after each addition of different concentrations of AA. F and F0 are the fluorescence intensities of N-CNPs in the presence and absence of AA, respectively. The insert is the parameters used in Equation 1. (C) Time-resolved fluorescence decay curves of N-CNPs in the absence (red curve) and presence (blue curve) of AA. Figure 3 (A) Fluorescence responses of N-CNPs upon addition of various concentrations of AA (from top to bottom, 0, 0.2, 0.4, 1, 2, 5, 10, 20, 30, 50, 80, 120 and 150 μM) in a pH 6 PBS solution. (B) Logarithmic value of F0/F of N-CNPs as a function of the concentration of AA. F0 and F are the fluorescence intensities of N-CNPs in the absence and presence of AA, respectively. Figure 4 (A) Fluorescence responses of N-CNPs towards several interferences. The black bars represent the addition of an excess of ions (5 mM for Na +, K+, Ca2+, NH4+, Mg2+, Cl-, F-, NO2-, and SO42-), glucose (5 mM), 50 μM for hydroquinone and GSH, 30 μM for DA and UA, 5 μM for ATP and NADH, 50 μg mL-1 for BSA and Hb to a 0.01 mg mL-1 solution of N-CNPs (pH 6.0). The white bars represent the subsequent addition of 50 μM AA to the solution. (B) Fluorescence of N-CNPs toward several amino acid. The black bars represent the addition of 50 μM amino acid to a 0.01 mg mL-1 solution 22


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of N-CNPs (pH 6.0). The white bars represent the subsequent addition of 50 μM AA to the solution. (C) Fluorescence response of N-CNPs upon addition of various concentrations of (a) AA, (b) D-isoascorbic acid, (c) catechol, and (d) dehydroascorbic acid in a pH 6.0 PBS solution. Figure 5 Fluorescence responses of N-CNPs in the presence of different concentrations of AA in human urine, human blood serum, and human blood plasma samples.

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Scheme 1

24


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Figure 1

100

C

B

20 0 4000

3000

1310 1570 1400

40

1250 1095

60

2000

N1s

Intensity (a.u.)

80

3400 3250

Transmitance (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


394

1000

396

398

400

402

Binding energy (eV)

Wavenumber (cm-1)

25


404


2.0

A

b

d

600 1.5 400 1.0

0.0 200

0 300 400 500 Wavelength (nm)

Normalized Intensity

0.5

200

c

a

1.0

600

Fluorescence Intensity (a.u.) F/F0

Figure 2

Absorbance (a. u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.9

B

0.8

0.7

0.6 0

5

10

C

0.5

0.0 20

15

20

25

Concentration of AA (M)

40

60

Time (ns)

26


80

30

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Figure 3 A

1.0

0

600

B

0.8

150 M

400

log (F0/F)

Fluorescence Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


200 0

0.6 0.4 0.2 0.0

400

450

500

550

600

-20 0

Wavelength (nm)

20 40 60 80 100 120 140 160 Concentration of AA (M)

27



Figure 4 A 1.0

0.8

0.8

F/F0

1.0

0.6 0.4

B

0.6 0.4 Pr o Le u G lu Al a Se r G ly Th r Ile G ln As p Ph e G ly Ly s M et Ty r Hi s Cy s Tr p

K+ N a+ C a 2+ M g 2+ N H + 4 C SO l 2 4 Fgl NO uc 2 os e H Q G SH A N TP A D DH A U A B SA H b

C

N-CNPs N-CNPs + 50 M sample N-CNPs + 100 M sample

HO

1.2

HO

HO

O

O

HO

HO

F/F0

F/F0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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OH

O

O

HO

HO

OH

O

HO

O

O

0.8 0.4 0.0 a

b

O

HO

HO

c

d

28


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Figure 5

2.0 1.5 1 .0

F0 /F

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0 .5 40

0 .0

30

CA A(

M

)

20

10

0

Blood plasma Blood serum Urine

29



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For TOC only

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Non-Redox Modulated Fluorescence Strategy for Sensitive and

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