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Highly Fe3+-selective fluorescent nanoprobe based on ultra-bright N/P co-doped carbon dots and its application in biological samples Jingfang Shangguan, Jin Huang, Dinggeng He, Xiaoxiao He, Kemin Wang, Runzhi Ye, Xue Yang, Taiping Qing, and Jinlu Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01053 • Publication Date (Web): 19 Jun 2017 Downloaded from http://pubs.acs.org on June 20, 2017

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Analytical Chemistry

Highly Fe3+-selective fluorescent nanoprobe based on ultrabright N/P co-doped carbon dots and its application in biological samples Jingfang Shangguan, Jin Huang, Dinggeng He, Xiaoxiao He,* Kemin Wang,* Runzhi Ye, Xue Yang, Taiping Qing, and Jinlu Tang State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, College of Chemistry and Chemical Engineering, Hunan University. Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, Changsha 410082, China * E-mail: [email protected]. Phone: +86-731-8882-3073 * E-mail: [email protected]. Phone: +86-731-8882-1566

ABSTRACT: Measuring the levels of Fe3+ in human body has attracted considerable attention for health monitoring as it plays an essential element in many physiological processes. In this work, we reported a selective fluorescent nanoprobe for Fe3+ detection in biological samples based on ultra-bright N/P co-doped carbon dots. By employing adenosine 5'-triphosphate (ATP) as the carbon, nitrogen and phosphorus source, the N/P co-doped carbon dots could be simply prepared through hydrothermal treatment. The obtained carbon dots exhibited high quantum yields up to 43.2%, as well as excellent photostability, low toxicity and water solubility. Due to the Fe-O-P bonds formed between Fe3+ and the N/P co-doped carbon dots, this nanoprobe showed highly selectivity towards Fe3+ against various potential interfering substances in the presence of EDTA. The fluorescence quenching of asfabricated carbon dots was observed as the increasing of Fe3+ concentration and the calibration curve displayed a wide linear region over the range of 1-150 µM with a detection limit of 0.33 µM. The satisfactory accuracy was further confirmed with the river samples and ferrous sulfate tablets, respectively. With the above outstanding properties, these N/P co-doped carbon dots were successfully applied for direct detection of Fe3+ in biological samples including human blood serum and living cells. Compared with the most reported carbon dots-based Fe3+ sensors, this nanoprobe showed high fluorescence, good accuracy and excellent selectivity, which presents the potential practical application for diagnosis of Fe3+ related disease.

Ferric ion (Fe3+), an essential element for living organism, plays important roles in many physiological actions including enzyme catalysis,1 oxygen transportation,2 electron transfer3 as well as hemoglobin synthesis.4 Both the deficiency and the accumulation of excess Fe3+ in human body can induce serious biological disorders and physiological damages, such as iron deficiency anemia (IDA),5 liver injury,6 renal failure,7 heart disease8 and cancer.9 Thus, the determination of Fe3+ in biological, medical, and environmental samples are of vital importance in human health monitoring. Currently, several analytical techniques including atomic absorption spectrometry (AAS),10 inductively coupled plasma mass spectrometry (ICP-MS)11 and electrochemical method12 have been established for Fe3+ quantification. However, most of the mentioned techniques require sophisticated instrumentations and complicated sample preparation procedures, which limit their wider applications. Fluorescent spectrometry, in contrast, has attracted considerable interest to meet the growing demand for rapid and sensitive Fe3+ detection as it possess excellent spatial and temporal resolution, high sensitivity, simplicity and fast response. Generally, the fabrication of fluorescent Fe3+ sensors can be achieved by combining a fluorophore moiety with an ionophore moiety, in which the ionophore moiety can selectively bind Fe3+ to form Fe-fluorophore complex and induce fluctuation in fluorescence intensity subsequently. Up

to date, various materials such as organic fluorescent dyes,13 gold nanoclusters,14 metal-organic frameworks (MOFs)15 and reduced graphene oxide (rGO),16 have been utilized as fluorescent sensors for Fe3+ monitoring. However, it still suffers from some drawbacks in the above fluorescent methods. For instance, some probes based on organic fluorescent dyes are not suitable for detection Fe3+ in living cells because of the toxicity, poor solubility and the rapid photobleaching. Some of the nanomaterial-based sensors need a relatively more complicated preparation and purification steps, which means incapability of simple and fast detection. Hence, it is of great value to develop novel fluorescent nanoprobes for Fe3+ quantification with low cytotoxicity, high selectivity and easy for synthesis. Carbon dots (CDs), as a new class of fluorescent carbon nanomaterials, have attracted tremendous attention due to their unique optical properties, highly photostability, non-toxicity and excellent biocompatibility. By virtue of these distinct benefits, much advance has been achieved in a variety of applications, such as sensing,17 bioimaging,18 drug delivery,19 ink,20 and optoelectronic devices.21 Therein, an increasing number of “on-off” probes for Fe3+ detection based on the fluorescent carbon dots have been explored.22-25 Different functionalized carbon dots can be used to fabricate various Fe3+ sensors, which have been find applications in aqueous

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analysis as well as bioimaging and biosensing both in vitro and in vivo.26-30 For example, Sun and the co-workers developed a carbon dots-based multifunctional biosensor for Fe3+ and pH detection in aqueous solution. This new approach was capable of selective detecting Fe3+ in river water according to the direct interaction between Fe3+ and the COOH and NH2 groups on the surface of the carbon dots.26 Chen et al. synthetized a nitrogen doped carbon dots for Fe3+ monitoring in living cells. Due to the formation of complexes between Fe3+ and the phenolic hydroxyl groups on the as-prepared carbon dots, the fluorescence intensity of this probe was Fe3+sensitive and could be used for sensing Fe3+ in Hela cells.27 However, to serving as a more promising substitute for quantification of Fe3+ for practical applications, there are some problems that impede their further bio-applications. First, the fluorescence quantum yields (QYs) of the most reported carbon dots is relatively low. Although it has been proved that the fluorescence quantum yields could be improved largely by heteroatom doping method, and intensive researches have been focused on the development of enhanced fluorescent carbon dots. Nevertheless, it was still not high enough in the most reported doped carbon dots (usually below 30%). Second, the specificity of some carbon dots-based Fe3+ sensing systems are not good enough due to the co-existing Ag+, Hg2+ , Pb2+ and Cu2+, which can also quench the fluorescence through the charge transfer process when they are close to the surface of the carbon dots. Finally, the efficacy and accuracy of the carbon dots for Fe3+ detection are unsatisfactory under the potential interfering substances in complex biological samples. For example, it is known that the content of serum iron is considered to be an important index for health monitoring. Unfortunately, there are only limited examples of carbon dots used for the direct and accurate evaluation of Fe3+ in such biological samples. Therefore, it is very important and encouraging to develop a new efficient platform based on carbon dots for direct Fe3+ detection in biological samples with strong fluorescence and distinguished selective properties. Towards this end, we synthesized the ultra-bright N/P codoped carbon dots from adenosine 5'-triphosphate (ATP) and further explored their use as a highly efficient Fe3+ nanoprobe. As shown in Scheme 1, N/P co-doped carbon dots were obtained through hydrothermal heating of ATP at 220 °C for 6 h, in which the ATP was used as the carbon, nitrogen and phosphorus source, simultaneously. The prepared carbon dots showed excellent fluorescence stability, low toxicity, and a high quantum yields up to 43.2% under the optimized preparation conditions. More importantly, it was demonstrated that the N/P co-doped carbon dots were highly selective towards Fe3+ in the presence of EDTA, in which the Fe-O-P bonds were formed by the coordination between Fe3+ and the Scheme 1. Schematic diagram for synthesizing highly fluorescent N/P co-doped carbon dots and their application in Fe3+ detection

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phosphate groups on the as-prepared carbon dots. Finally, this carbon dots-based nanoprobe was employed for assaying Fe3+ in aqueous solution samples and biological samples with satisfactory results, which exhibited practical potential for various bio-applications.

EXPERIMENTAL SECTION Materials. Adenosine 5'-triphosphate (ATP), adenosine-5'diphosphate (ADP), adenosine-5'-monophosphate (AMP), adenosine, quinine sulfate (98 %, suitable for fluorescence) and ethylenediaminetetraacetic acid tetrasodium salt (EDTA•2Na) were purchased from Sangon Biotechnology Inc. (Shanghai, China). Dimethyl sulfoxide (DMSO) was obtained from Xilong Reagents Company (Guangdong, China). NaCl, KCl, AgNO3, CaCl2, ZnCl2, MgCl2•6H2O, CuSO4•5H2O, MnSO4•H2O, CoSO4•7H2O, NiSO4•6H2O, Hg(NO3)2, Pb(NO3)2, AlCl3, CrCl3, FeCl3 and ZrOCl2•8H2O were obtained from Sinopharm Chemical Rea-gent Co,. Ltd (Shanghai, China). Ferrous sulfate tablets were purchased from Yongning pharmaceutical Limited by Share Ltd (Ji’nan, China). All solutions were prepared and diluted by deionized water from the Milli-Q ultrapure water system. Preparation of N/P co-doped carbon dots and N doped carbon dots. We synthesized the N/P co-doped carbon dots by the reported hydrothermal method with a slight change 31. Briefly, 1 g of ATP was dissolved with 30 mL ultrapure water. Then the obtained solution was placed in a 50 mL Teflonsealed autoclave reactor and heated at 220 °C for 6 h. After cooling down to room temperature, the products were purified using a 0.22 µm filtration membrane to remove nonfluorescent deposits and then dialyzed against ultrapure water through a dialysis membrane (1000 Kd) for 48 h. The asprepared N/P co-doped carbon dots solution was stored at 4 °C. In order to prepare the N/P co-doped carbon dots with high quantum yields, the hydrothermal conditions were varied and optimized, during which the synthesis temperature was changed from 160 to 220 °C, and the reaction time was varied from 2 to 8 h. Additionally, using 0.78 g of ADP and 0.60 g of AMP as precursor respectively, N/P co-doped carbon dots with different phosphorus doping content were synthesized. For synthesis of N doped carbon dots, 0.44 g of adenosine was used as precursor and the same procedures described above were carried out. Quantification of Fe3+ in aqueous solution. The detection of Fe3+ was performed in aqueous solution at room temperature. In a typical assay, standard stock solutions of Fe3+ with different concentrations were first prepared by dissolving FeCl3 in deionized water. Subsequently, 80 µL of solution containing 0.1 mg mL-1 N/P co-doped carbon dots was mixed with 10 µL of different concentration of Fe3+ solutions and 10 µL of 25 mM EDTA solution. The fluorescence emission spectra before and after the addition of Fe3+ were recorded with excitation wavelength fixed at 320 nm, respectively. The application of Fe3+ detection in real samples were carried out using river water and ferrous sulfate tablets as the analyzed samples. In briefly, river samples were collected from Xiangjiang River (Changsha, China) followed by filtered through a 0.22 µm membrane (Millipore) to remove the large particles before use. Then, 70 µL of carbon dots solution was mixed with 10 µL of river sample, 10 µL of 25 mM EDTA

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solution and 10 µL of different concentration of Fe3+ solution. The content of the added Fe3+ in river samples was analyzed using the developed sensing technique and the recovery efficiency was calculated. For quantification of Fe3+ in ferrous sulfate tablets, a piece of ferrous sulfate tablets was dissolved in 50 mL of dilute nitric acid. After heated and evaporated twice, the products were finally dissolved in 50 mL of deionized water and stored at 4 °C before use. Detection of Fe3+ in human serum. The human serum was first treated to release the Fe3+ from proteins. Briefly, equal volume of human serum and anhydrous ethanol were mixed and heated at 95°C for 15 min. After cooling down to room temperature, the serum solution was sonicated for 2 min. Then, the protein precipitate was removed by centrifugation and the supernatant was collected for further use. In the analytical assay, 0.1 mg of N/P co-doped carbon dots was dissolved with 10 µL of 25 mM EDTA and different volumes of above deproteinized human serum (0, 20, 40, 60, 80, 100 µL), and then diluted to 110 µL with deionized water. The resulting solution was incubated at room temperature for 30 min and analyzed with the proposed method. For quantification of the unknown amount of Fe3+ in human serum, the standard addition method with FeCl3 as the standard was performed. The deproteinized human serum samples were first spiked with Fe3+ at different concentrations levels (5 µM, 10 µM, 15 µM, 20 µM, 25 µM,) and then measured using the N/P co-doped carbon dots. In the test solutions, the human serum was diluted 4 times. After incubation, the fluorescence emission of the samples at 408 nm was recorded. Intracellular Fe3+ Sensing. Hela cells were grown in coverglass bottom dishes (35 mm × 10 mm) for 12 h. Then, the cells were incubated for 3 h in culture medium containing 0.25 mg mL-1 N/P co-doped carbon dots. Before use, the cells were washed with 1 mL of phosphate buffer solution (PBS, 10 mM, pH 7.4) for three times to remove the excessive carbon dots. The treated cells were then incubated with 150 µM of FeCl3 solution in medium for another 2 h and washed with PBS buffer. The cell images were observed under a confocal fluorescence microscope with excitation fixed at 405 nm.

RESULTS AND DISCUSSION Preparation of ultra-bright fluorescent carbon dots. The N/P co-doped fluorescent carbon dots were obtained by hydrothermal treatment of adenosine 5'-triphosphate (ATP). Moreover, a control experiment was also performed using adenosine as the only precursor to synthetize N doped fluorescent carbon dots. The synthesis conditions including hydrothermal temperature and reaction time were changed to study their effect on the optical property of the carbon dots. Using quinine sulfate as a reference, the quantum yields of the prepared N/P co-doped carbon dots and N doped carbon dots were calculated and depicted in Table S1. It was described that the quantum yields of the N/P co-doped and the N doped carbon dots were both increased as the temperature change from 160 to 220 °C. When heated at 220 °C, with the prolonging of reaction time from 2 to 8 h, they both displayed a maximum value at 6 h. As shown in Table S1, the quantum yields of N/P co-doped carbon dots was as high as 43.2 %

Figure 1. Characterization of N/P co-doped carbon dots. (A) The HRTEM image. (B) The FTIR spectrum of the ATP (line a) and the N/P co-doped carbon dots (line b). (C) XPS survey scan of N/P co-doped carbon dots. (D) High-resolution of C 1s peaks. (E) High-resolution of N 1s peaks and (F) High-resolution of P 2p peaks.

under the optimized conditions (220 °C, 6 h) while the N doped carbon dots only have 29.0 % under the same conditions. Up to now, it has been reported that various heteroatoms, such as boron (B),32 nitrogen (N),33 sulfur (S),34 silicon (Si)35 and phosphorus (P), 36 could be used as doped elements and influence the structural properties and the photoluminescence performance of prepared carbon dots. The above results confirmed that the N/P-co doped carbon dots have a higher fluorescence quantum yields compared with that of N doped carbon dots. This phenomenon was possibly resulted from the N/P co-doping induced modulation of the chemical and electronic characteristics of the carbon dots. To the best of our knowledge, the quantum yields of the N/P codoped carbon dots is much higher than most of the reported doped carbon dots,20-39 which is remarkable and excellent, notably compared with other reported carbon dots-based Fe3+ sensors. (The survey result was shown in Table S2). Thus, it was indicated that the fabrication of highly fluorescent carbon dots could be easily achieved using ATP as the precursor. Characterization of the N/P co-doped carbon dots. The morphology of the obtained carbon dots was investigated using higher resolution transmission electromicroscope (HRTEM) and ZetaSizer Nano, respectively. Figure 1A depicted the HRTEM image of the carbon dots, revealing that they were spherical in shape and dispersed without apparent aggregation. The diameters of N/P co-doped carbon dots were also investigated by dynamic light scattering measurement, which showed a narrow range from 4.2-8.7 nm (Figure S1A). The crystal phase of the carbon dots was further confirmed by the X-ray diffraction (XRD) analysis. As shown in Figure S1B, the diffraction pattern has a broad, very low-intensity

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Figure 2. Optical properties of N/P co-doped carbon dots. (A) UV-vis absorption and fluorescence spectrum of N/P co-doped carbon dots. The inset is the photographs of the carbon dots under visible light (left) and 365 nm UV light (right). (B) Fluorescence emission spectra of as-prepared carbon dots at different excitation wavelength. diffraction peak centered at 25.8°, which demonstrated the amorphous structure of the carbon dots. Surface analysis was carried out by FT-IR spectra and X-ray photoelectron spectroscopy (XPS). As illustrated in Figure 1B, the FT-IR spectra of adenosine 5'-triphosphate (ATP) and the assynthesized carbon dots were investigated. The results revealed that the carbon dots exhibiting the typical peaks at 583 cm-1, 906 cm-1 and 1096 cm-1 were contributed to the P-O and P=O, which showed the same peak position compared with the ATP (Figure 1B, line a). Peaks at 1406 cm−1 , 1643 cm−1, 3150 cm−1 , and 3350 cm−1 suggested the existence of the C-N stretching vibrations, C=O stretching mode, N-H and O-H, respectively. The XPS was used to determine the surface composition and elemental oxidation state of the carbon dots. The element identification in Figure 1C confirmed that there were four elements existed in the N/P co-doped carbon dots, 40.21 % of carbon, 39.11 % of oxygen, 11.59 % of nitrogen and 9.09 % of phosphorus. The high-resolution XPS survey scan of C 1s could be deconvoluted into five contributions, corresponding to C=C at binding energy of 284.3 eV, C-C at 284.9 eV, C-N at 285.7 eV, C-O at 286.2 eV and C=O at 288.2 eV. The high-resolution N 1s spectra (Figure 1E) at binding energy of 398.9 eV, 399.8 eV and 400.6 eV could be easily identified, which were attributed to the formation of C=N-H, N-H and C-N, respectively. The XPS spectrum of P 2p exhibited two apparent peaks centered at 133.1 eV and 133.7 eV, which were related to the P-O and P=O bond (Figure 1F). The surface components of the N/P co-doped carbon dots characterized by XPS were in agreement with the FT-IR analysis, which suggesting the successful synthesis of N/P co-doped carbon dots.

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Figure 3. The photostability of as-synthesized carbon dots. (A) Fluorescence stability studies of N/P co-doped carbon dots in different pH solutions. (B) Fluorescent time scan of N/P codoped carbon dots for 2 h under 320 nm excitation. The UV-vis absorption and fluorescence emission spectrum studies were carried out to further explore the optical properties of as-prepared carbon dots. As shown in Figure. 2A. The N/Pco-doped carbon dots exhibited a bright blue fluorescence under 365 nm irradiation (inset), and displayed no obvious absorption peak under 500 nm. Then, a detail investigation of the fluorescence property was performed. With the regular increase of excitation wavelength from 300 to 400 nm, the fluorescence emission of N/P co-doped carbon dots showed an excitation-dependent behaviour. It exhibited an intensity maximum at 408 nm under 320 nm excitation (Figure 2B). The stability of N/P co-doped carbon dots. When the carbon dots were used for practical sensing applications, a good water solubility and fluorescence stability were quite necessary. The prepared N/P co-doped carbon dots were inherently water soluble as there are lots of oxygen-rich functional groups in the structures, as it was already proved by the XPS and FT-IR analysis. To investigate the fluorescence stability, the influences of various conditions on the carbon dots were studied. As shown in Figure 3A, no significant change of the fluorescence intensity in a solution with pH values ranging from 3.0 to 12.0 was observed, indicating the very stable property of the carbon dots even under extreme pH conditions. Time scan of the fluorescence intensity was also carried out under 320 nm excitation for 2 h. It was clearly demonstrated that the fluorescence intensity remained strong and stable during the scanning process (Figure 3B). In order to realize the application of the nanoprobe in more complex biological samples, several typical amino acids and different concentrations of ATP in the biological system were used as

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Figure 4. Sensitive detection of Fe3+ using N/P co-doped carbon dots. (A) Fluorescence intensity response of N/P co-doped carbon dots in the presence of 2.5 mM EDTA and an increasing concentration of Fe3+: 0, 0.1, 0.5, 1, 5, 10, 20, 40, 60, 80, 100, 150, 200, 250, 300, 400 and 500 µM. (B) The linear relationship of (F0-F)/F0 versus the concentration of Fe3+ over the range of 1-150 µM.

potential interferences to test their fluorescence stability. As shown in Figure S2, the change of fluorescence intensity ratios (F/F0) under these amino acids (Ala, L-Arg, L-Cys, LGSH,Lys and L-Ser) and a high concentration of ATP (up to 1000 µM) were negligible. Therefore, it was confirmed that the obtained N/P co-doped carbon dots possess excellent fluorescence stability under different conditions. Fluorescent response property of N/P co-doped carbon dots towards Fe3+. Taking the advantages above, N/P codoped carbon dots were utilized as a fluorescent sensor for sensitive and selective detection of Fe3+. Figure 4A depicted the change of fluorescence intensity of the as-synthetized carbon dots in the presence of 2.5 mM of EDTA and different concentration of Fe3+ (0, 0.1, 0.5, 1, 5, 10, 20, 40, 60, 80, 100, 150, 200, 250, 300, 400 and 500 µM). It clearly demonstrated that the fluorescence could be quenched gradually with the increasing concentration of Fe3+. As shown in Figure 4B, the quenching efficiency ((F0-F)/F0) displayed a good linear relationship (R2 = 0.9974) versus the concentration of Fe3+ in a wild range of 1-150 µM, where F0 and F presented the fluorescence intensity at 408 nm in the absence and presence of Fe3+, respectively. The limit of detection (LOD) was estimated to be 0.33 µM, which was calculated according to a signal-to-noise ratio of S/N = 3. The LOD presented by our method was much lower than the limit of Fe3+ in drinking water (~ 5.357 µM) set by U. S. Environmental Protection Agency,40 indicating that the carbon dots could be applied as an efficient nanoprobe for Fe3+ detection.

Figure 5. (A) Ion selectivity studies of the N/P co-doped carbon dots in the presence of EDTA. The black bar represents the fluorescence intensity ratios (F/F0) of the competing metal ions to the as-prepared carbon dots. The grey bar represents the changed values of fluorescence intensity ratios that occurred upon addition of Fe3+ ions to the previous solution. (B) The fluorescence intensity ratios (F/F0) of various metal ions to the carbon dots in the absence (black bar) and in the presence (grey bar) of EDTA. (The final concentration of Na+, K+, Ag+, Ca2+, Mg2+, Zn2+, Cu2+, Co2+, Cd2+, Mn2+, Pb2+, Hg2+, Al3+, Fe3+ Cr3+ and Zr4+ was 300 µM and the EDTA was 2.5 mM).

Selectivity study. The complexity of real samples presents a great challenge to the carbon dots for metal ion detection not only in sensitivity but more importantly in selectivity. Thus, the selectivity studies were carried out to evaluate the performance of the proposed nanoprobe. Different metal ions including Na+, K+, Ag+, Ca2+, Mg2+, Zn2+, Cu2+, Co2+, Cd2+, Mn2+, Pb2+, Hg2+,Al3+, Cr3+ and Zr4+ were chosen to assess their impact on the fluorescence emission of N/P co-doped carbon dots under same conditions. The black bar in Figure 5A depicted the fluorescence intensity ratios (F/F0) of N/P codoped carbon dots in the presence of EDTA and the metal ions. It was showed that no significant decrease in fluorescence intensity could be observed with these fifteen ions. Subsequently, a certain concentration of Fe3+ was added to the above solutions to form a competing mixture and the fluctuation of the fluorescence intensity ratios (F/F0) was recorded. As shown in grey bar of Figure 5A, a similar value of the fluorescence intensity ratios (F/F0) was obtained after the solution added with Fe3+. More importantly, it was consistent with the control group, in which the carbon dots were mixed with Fe3+ merely. The results indicated the remarkable selectivity of the N/P co-doped carbon dots for Fe3+. According to the above achievements, we speculated that this outstanding selectivity could probably be attributed to the existence of EDTA, by which the fluorescence emission was

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quenched by Fe3+ due to the strong binding preference of Fe3+ towards the phosphate group on the surface of the N/P codoped carbon dots, while the other metal ions were chelated by EDTA. To prove this speculation, the fluorescence intensity ratios (F/F0) under representative metal ions in the absence and presence of EDTA were investigated. As shown in black bar of Figure 5B, besides Fe3+, several metal ions could also quench the fluorescence of the carbon dots without the existence of EDTA, such as Ag+, Cu2+, Co2+, Pb2+, Zr4+ and Hg2+. However, in the presence of EDTA, the values of fluorescence intensity ratios (F/F0) were increased in all metal ion groups expect that of Fe3+ and the fluorescence quenching efficiency of all these interfering ions displayed no significant difference with the control group under the chelation of EDTA. Thus, it is clearly illustrated that the as-synthesized carbon dots displayed highly selective response to Fe3+ under the masking effect of EDTA. In addition, the Fe3+ selective verification was carried out by N doped carbon dots and the N/P co-doped carbon dots with different phosphorus doping content. As displayed in Figure S3, the fluorescence intensity of N doped carbon dots only has a slight decrease by Fe3+ in the presence of EDTA (Figure S3A), while remarkably quenching effect was observed in the same measuring condition of N/P co-doped carbon dots. The quenching effect (F/F0) of different N/P co-doped carbon dots for Fe3+ were 0.36 for AMP-CDs (Figure S3B), 0.35 for ADPCDs (Figure S3C) and 0.31 for ATP-CDs (Figure S3D), respectively. These results revealed that the sensing capability and selectivity property were strongly depended on the functional phosphorus groups and the fluorescence intensity of N/P co-doped carbon dots synthesized from ATP exhibited more sensitive towards Fe3+ when compared with AMP-CDs and ADP-CDs. Possible quenching mechanism. In general, fluorescence quenching process refers to the interaction between the fluorescent molecules and the quencher molecules, which usually originates from dynamic or static quenching. The quenching constant can be determined by standard SternVolmer Eq. (1) that provide a measure of the binding affinity between fluorescent molecules and the quencher molecules. (1) F0 / F = 1 + ksv/ [Q] Where F0 and F refer to the fluorescence intensity in the absence (only N/P co-doped carbon dots) and presence of quencher (Fe3+), respectively. [Q] represents the concentration of the quencher, ksv is the quenching constant. It was confirmed that the fluorescence intensity of N/P codoped carbon dots could be quenched significantly by Fe3+. To explore the possible quenching mechanism, the dependence of fluorescence quenching on Fe3+ were analyzed by the SternVolmer equation. As shown in Figure S4A, the Stern-Volmer plot followed a good linear trend over the Fe3+ concentration range from 1-150 µM. The Stern-Volmer quenching constant (ksv) was obtained by EQ. (1) as 5.3 × 103 M-1. Since the ksv value is large, we speculated that it may be due to static quenching, which refers to the formation of non-fluorescent complexes by combining ground-state fluorescence molecules with quencher molecules. The dynamic or static quenching can also be discriminated by calculating the change of quenching constant value under different temperatures. As depicted in Figure S4B, the Stern-Volmer quenching constant

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at 318 K was 5.2 × 103 M-1, which was found to be slightly less than that of at 298 K (5.3 × 103 M-1). This result suggested that Fe3+ was not dissociating from N/P co-doped carbon dots even at 318 K. The fluorescence lifetime study is one of most useful method to distinguish dynamic and static quenching. In order to get deep inside of the quenching mechanism, the fluorescence lifetime investigation of N/P co-doped carbon dots with or without Fe3+ was carried out. After addition of Fe3+, the average fluorescence lifetime of the N/P co-doped carbon dots exhibited nearly constant lifetime value from 5.87 ns to 5.85 ns (Figure S4C), indicating the fluorescence quenching provoked by Fe3+ was probably due to static quenching arising from the formation of Fe-O-P bond. To further confirm this deduction, the FT-IR and zeta-potential analysis were performed to prove that Fe3+ indeed complexed with the surface functional groups of N/P co-doped carbon dots. As depicted in Figure S5A, evident changes were observed at the characteristic peaks of P-O and P=O group region (906 cm-1 and 1130 cm-1). Meanwhile, after addition of Fe3+, the zeta potential value was changed from -13.8 mV to + 11.3 mV, which demonstrated that Fe3+ was selectively coordinated with the negative phosphorus group on the asprepared carbon dots (Figure S5B). Accuracy investigation. In view of the highly selectivity and sensitivity Fe3+ detection abilities of the N/P co-doped carbon dots, the accuracy of this proposed nanoprobe was further confirmed by detecting Fe3+ in river samples and ferrous sulfate tablets. First, the accuracy was tested using practical water samples with a standard addition technique. The synthetic water samples were collected from Xiangjiang River (Changsha, China) and spiked with different concentrations of Fe3+. As listed in Table S3, the detection efficiencies of Fe3+ in spiked water samples varied from 84.5 % to 94.0 %, revealing a reliable and practicable method for Fe3+ detection. Then, the investigation of Fe3+ measurement in ferrous sulfate tablets was carried out. The samples were pretreated with diluted nitric acid and the concentration of Fe3+ was detected using N/P co-doped carbon dots-based nanoprobe and atomic absorption spectrometry (AAS), respectively. As shown in Table S4, the results achieved by the proposed method were comparable with that of AAS analysis, suggesting the N/P codoped carbon dots could be utilized for selective and accurate quantification of Fe3+ in real samples.

Figure 6. The linear calibration plot for Fe3+ detection in human blood serum

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Table 1. Comparing of sensing performance of different carbon dots-based fluorescence sensors for Fe3+ detection Carbon dots

QYs

Anti-interference ion

Real sample

Quantitative detection

Ref

N-doped

31%

Ag+, Cu2+, Hg2+, Co2+, Ni2+

urine, serum

recovery

27

S-doped

10.6%

Ag+, Cu2+, Hg2+, Pb2+, Ni2+

1% serum

original Fe3+ content

35

N-doped

34.8%

Cu2+, Hg2+, Pb2+, Co2+

spring water

recovery

28

B, N, S co-doped

5.4%

Cu2+, Hg2+, Ni2+, Co2+

1% urine, 1% serum

recovery

30

N-doped

18.4%

Ag+, Hg2+, Co2+,Ni2+

Heme capsules


18

N-doped

30.7%

Cu2+, Hg2+, Pb2+, Co2+, Ni2+

river sample


26

N, P co-doped

43.2%

Ag+, Cu2+, Hg2+, Pb2+, Co2+,Ni2+

river sample, ferrous sulfate tablets, 25% serum

recovery and original Fe3+ content

This work

The anti-interference ion study in this table was focused on six ions (Ag+, Cu2+, Hg2+, Pb2+, Co2+, Ni2+), which may have great influence on the fluorescence of carbon dots.

Monitoring Fe3+ level in human serum. Taking into account these desirable properties of as-prepared carbon dots, the practical evaluation of this nanoprobe was carried out in human serum. As shown in Figure S5, the fluorescence intensity ratios (F/F0) was decreased and has a linear relation against the volume of human serum sample. This revealed that the proposed method was capable of monitoring Fe3+ in human serum sample. For quantification of the unknown concentration of Fe3+ in the commercial human blood serum, a standard addition method using FeCl3 as the standard was carried out (Figure 6). The concentration of Fe3+ in the serum was found to be 19.6 ± 0.7 µM, which was well consistent with the result (20.6 ± 0.4 µM) obtained by atomic absorption spectrometry (AAS). Compared with other carbon dots-based Fe3+ sensors, this nanoprobe was able to accurate detect the original Fe3+ content in biological sample instead of the recovery measurement of additional added Fe3+ due to the distinguished selective property and excellent fluorescence, which showed remarkable practicability for Fe3+ detection in real sample (Table 1). Sensing Fe3+ in living cells. After demonstrating the capability of the N/P co-doped carbon dots for selective and accurate Fe3+ detection in human serum, the ability of biosensing performance of the prepared nanoprobe for Fe3+ in living cells was further investigated using a confocal laser scanning microscope (CLSM). As depicted in the middle column of Figure S7A, the cellular uptake of N/P co-doped carbon dots for 2 h was monitored and recorded. As expected, it can be seen that the carbon dots incubated Hela cells displayed brightly fluorescence under the excitation of 405 nm, proving the abilities of these carbon dots to penetrate into the cell membrane. However, the fluorescence emission was almost quenched after Hela cells incubated with 150 µM of Fe3+ subsequently, confirming the ability of the N/P co-doped carbon dots for sensing intracellular Fe3+. In addition, the toxicity of the carbon dots was assessed using a standard MTT assay (Figure S7B). The cells viability was estimated to be greater than 80% after the Hela cells incubated with the carbon dots over a wide concentration range of from 0.01 to 0.25 mg

mL-1. The observations described herein well demonstrate that these ultra-bright fluorescent carbon dots have low toxic effect on cells and could serve as a promising candidate for intracellular Fe3+ sensing.

CONCLUSION To summery, we developed the N/P co-doped carbon dots as ultra-bright fluorescent nanoprobe for selective and sensitive detection of Fe3+. The carbon dots could be obtained via onepot hydrothermal reaction using ATP as the carbon, nitrogen and phosphorus source, simultaneously. The synthesized N/P co-doped carbon dots displayed highly photostability, low toxicity and respectable quantum yields up to 43.2%. The fluorescence intensity of as-prepared carbon dots was sensitive towards Fe3+ in a wild linear range from 1 to 150 µM with a limit of detection of 0.33 µM. Moreover, the carbon dotsbased nanoprobe demonstrated a high affinity to Fe3+ through Fe-O-P bonds. In the presence of EDTA, the N/P co-doped carbon dots exhibited highly Fe3+-selective property under various potential interfering substances and were successfully applied for Fe3+ detection with satisfactory results in aqueous sample solutions, as well as in complex matrix of biological samples. In comparison with other carbon dots-based Fe3+ sensors, our nanoprobe possess strong fluorescence, excellent anti-interference ability and the capability for direct Fe3+ quantification in biological samples. Thus, we believe that this N/P co-doped carbon dots can be exploited for Fe3+ monitoring in more biological applications, particularly in clinical diagnostics.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional information as noted in text. Experimental details, DLS and XRD analysis, fluorescence stability of N/P co-doped carbon dots in the presence of various amino acids and different concentration of ATP, fluorescent Fe3+ response of N doped

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carbon dots and and N/P co-doped carbon dots with different phosphorus doping content, Stern-Volmer plot analysis and fluorescence lifetime studies, The FT-IR and zeta-potential analysis of N/P co-doped carbon dots before (black line) and after (red line) the addition of Fe3+, the relationship between fluorescence intensity ratios (F/F0) of N/P co-doped carbon dots and the volume of deproteinized human serum added, cellular imaging and cytotoxicity studies, fluorescence quantum yields (QYs) of doped carbon dots under different hydrothermal conditions, fluorescence quantum yields of heteroatom doped carbon dots from different reactants, detection of Fe3+ in the spiked water samples, detection of Fe3+ in ferrous sulfate tablets. (PDF)

Corresponding Author * E-mail: [email protected]. Phone: +86-731-8882-3073; Fax: 86-731-88821566; * E-mail: [email protected]. Phone: +86-731-8882-1566; Fax: 86-731-8882-1566;

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported in part by the Key Project of Natural Science Foundation of China (Grants 21190044, 21221003 and 21675046).

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