Carbon Dots Enhance the Nitrogen Fixation Activity of Azotobacter

Apr 27, 2018 - application in biosensing technology,10−12 biomedical engineer- ing,10 and ... by online GC-7900 gas chromatograph (GC) with a therma...
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Carbon Dots Enhance the Nitrogen Fixation Activity of Azotobacter Chroococcum Huibo Wang,†,§ Hao Li,†,§ Mengling Zhang,† Yuxiang Song,† Jian Huang,‡ Hui Huang,† Mingwang Shao,† Yang Liu,*,† and Zhenhui Kang*,† †

Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Institute of Functional Nano & Soft Materials (FUNSOM) and ‡School of Biology & Basic Medical Sciences, Soochow University, 199 Ren’ai Road, Suzhou 215123, Jiangsu, P. R. China S Supporting Information *

ABSTRACT: Biological nitrogen fixation is critical for the nitrogen cycle on the earth. Nitrogen-fixing bacteria, as an environmentally friendly microorganism, convert atmospheric nitrogen to available nitrogen source for plants. In this study, we found that carbon dots (CDs) could significantly enhance the nitrogen-fixing activity of azotobacter chroococcum, in which the activity of azotobacter treated with CDs (4 μg/mL) was increased by 158% compared to that of the control one. A series of experiments suggest that CDs can combine with the nitrogenase, affect the secondary structure of nitrogenase, improve the electron transfer in the biocatalytic process, and finally improve nitrogenase activity for nitrogen fixation. Our findings may offer an economical and environmentally friendly means of improving the biological nitrogen fixation as well as solving the insufficiency of nitrogen fertilizer. KEYWORDS: carbon dots, biological nitrogen fixation, azotobacter chroococcum, nitrogenase, nitrogen fixation activity

1. INTRODUCTION Nitrogen is the most abundant element in the atmosphere, mainly in the form of nitrogen gas. However, most plants and animals cannot utilize nitrogen gas directly and only some microorganisms, such as nitrogen-fixing bacteria, are able to metabolize nitrogen by biological nitrogen fixation. Biological nitrogen fixation is a key part of the nitrogen cycle, converting atmospheric nitrogen (N2) to ammonia (NH3) with the existence of nitrogenase.1−3 In agriculture, crops need to absorb a large amount of nitrogen nutrition in soil as they grow and thus the soil loses a lot of nitrogen every year.4 To meet the demand for nitrogen fertilizer, it is vital to improve the nitrogen fixation activity for nitrogen-fixing bacteria, and various techniques have been exploited. The rapid development of nanotechnology motivates researchers’ interest to explore the potential effects of nanomaterials on biological nitrogen fixation, which may provide an effective technique to solve the problem of nitrogen shortage. Carbon dots (CDs), as a unique and promising carbon-based nanomaterial, have good water solubility, easy modification, tunable fluorescent properties, and biocompatibility.5−9 These advantages of CDs provide more opportunities for their application in biosensing technology,10−12 biomedical engineering,10 and bioimaging,13−15 ranging from the detection of heavy metal ions, drug/gene delivery, and cellular imaging. Recently, some reports suggested that CDs could serve as a novel regulator for plant growth as well as good biocompatibility, which exhibited the potential value of CDs in agriculture.16−18 © XXXX American Chemical Society

Lei et al. found that CDs could be uptaken and transported into plant cells and regulate the growth of mung bean sprouts.18 Holden et al. found that carbon nanotubes could improve the nitrogen fixation potential of soybean in soil.19 In addition, Han et al. reported that carbon nanotubes could promote nodulation in the rhizobium-legume system.20 Yang et al. reported that CDs also have been used in the microorganism field, which inspired us to study the interaction between CDs and bacteria.21 Nevertheless, there are a very limited number of reports on the potential impact of CDs on the nitrogen-fixing bacteria in the biological nitrogen fixation field. In this study, we have investigated the effect of CDs on the nitrogen-fixing bacteria and proposed the possible role of CDs in biological nitrogen fixation. Azotobacter chroococcum, as a nitrogen-fixing bacterium, was selected as the model for studying the impact of CDs on biological nitrogen fixation.3,22 We have evaluated the effects of CDs on the nitrogen fixation activity of azotobacter. The nitrogen-fixing activity of azotobacter treated with CDs (4 μg/mL) was increased by 158% compared to the control. To understand how CDs affect the nitrogen fixation of azotobacter, separate studies of CDs on the growth of azotobacter, nitrogenase activity, and nitrogenase structure were performed. First, CDs have a dose−response effect on the growth of azotobacter, and the presence of CDs (4 Received: March 6, 2018 Accepted: April 27, 2018

A

DOI: 10.1021/acsami.8b03758 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. (a) TEM image of CDs; inset is the HRTEM of CDs. (b) Size distribution, (c) FTIR spectrum (red line) and UV−vis spectrum (black line), and (d) PL spectra of CDs. (e) Full XPS spectrum. (f) High resolution of C 1s spectrum for CDs.

μg/mL) could promote the growth of azotobacter significantly. The following investigations indicate that the activity of nitrogenase/CDs was higher than that of free nitrogenase (increased by 56%). Spectroscopy analysis shows that the addition of CDs could affect the secondary structure of nitrogenase, especially the α-helix. In a previous report, CDs with electron-donating and electron-accepting properties could accelerate the electron transfer among the active sites of enzymes and improve their activity. In this study, CDs play a similar role in the catalytic reaction of nitrogen fixation, which could combine with nitrogenase, influence the structure of the enzyme, improve electron transfer, and enhance nitrogenase activity. Owing to these observations, CDs can be a good candidate for the improvement of nitrogen fixation and show great potential in agriculture.

spectroscopy (XPS) spectra were recorded with a KRATOS Axis ultra-DLD X-ray photoelectron spectrometer with a monochromatized Mg Kα X-ray (hν = 1283.3 eV). The UV−vis absorption spectrum was measured by a Lambda 750 (PerkinElmer) spectrophotometer. Fourier transform infrared (FTIR) spectrum was recorded on a Varian Spectrum GX spectrometer. Photoluminescence (PL) spectrophotometry was performed using a FluoroMax-4 (Horiba Jobin Yvon) spectrophotometer. Gas chromatography (GC) was estimated by online GC-7900 gas chromatograph (GC) with a thermal conductivity detector and 5 Å molecular sieve columns. 2.3. Synthesis of CDs. The carbon dots were prepared by an electrochemical method previously reported.23 2.4. Nitrogen-Fixing Bacteria Culture Condition. Azotobacter were supplied by the medium supplemented with 0.2 g/L KH2PO4, 0.8 g/L K2HPO4, 0.2 g/L MgSO4·7H2O, 0.1 g/L CaSO4·2H2O, 20 g/L mannitol, 0.5 g/L yeast extract, 15 g/L agar, FeCl3·6H2O (trace), Na2MoO4·2H2O (trace), and 1 L of ultrapure water for solid medium and without agar for liquid medium. Before incubation, the medium was sterilized by autoclaving at 120 °C for 20 min. After cooling to 60 °C, CDs were added to the medium. Next, azotobacter were incubated with different concentrations of CDs at 28 °C in the constanttemperature incubator. 2.5. Nitrogen Fixation Activity by Acetylene Reduction Assay (ARA). Nitrogen fixation assay was determined by acetylene reduction assay. The acetylene reduction assay (ARA) is a sensitive tool for monitoring the N2 fixation activity using acetylene as a

2. EXPERIMENTAL SECTION 2.1. Materials. All other chemicals were purchased from SigmaAldrich and Adamas-bate, and all chemicals were analytical grade. 2.2. Characterization. An FEI/Philips Tecnai G2 F20 TWIN transmission electron microscope operating at an acceleration voltage of 200 kV was taken to obtain high-resolution transmission electron microscopy (HRTEM) images. The atomic force microscopy (AFM) image was carried out by Veeco AFM. X-ray photoelectron B

DOI: 10.1021/acsami.8b03758 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 2. N2 fixation activity of azotobacter incubated with CDs of different concentrations (0, 2, 4, 6, 8, and 10 μg/mL). (a) Ratio of C2H4/C2H2, representing the N2 fixation activity measured by acetylene reduction assay. (b) Content of NH4+−N of azotobacter. All of the experiments were repeated three times at least. Marked with (*p < 0.05), (**p < 0.01), and (***p < 0.001) exhibit significant differences from the control. (* is the significance level, p is the p-value). nm.28 The experiments were repeated at least three times to acquire accurate data.

substrate catalyzed by nitrogenase. The chromatographic conditions were as follows: oven temperature 30 °C, injection port temperature 180 °C, and detector temperature 70 °C. The carrier gas was argon (Ar) with a flow rate of 30 mL/min. Before the experiments, the azotobacter were placed in closed serum bottles filled with air and acetylene (0.1 atm). After incubation at 28 °C for 24 h, the gas in these bottles was injected into gas chromatography. The N2 fixation activity could be evaluated by the decreased amount of acetylene and the increased amount of ethylene. The ratio of C2H4/C2H2 is used to evaluate the N2 fixation activity of different samples. The measurement of N2 fixation activity for nitrogen-fixing bacteria and nitrogenase was carried out by ARA with C2H2 as the substrate, just the procedures were different.24,25 2.6. Nitrogenase Activity Assay. Nitrogenase activity was measured by ARA. Nitrogenase was extracted as previously reported.26 Before experiments, the nitrogenase solution was saturated with Ar for at least 30 min. The optimal enzyme reaction condition took place in a sealed bottle containing 0.4 mmol/mL phosphate buffer (pH = 7.4), 0.2 mmol/mL MgCl2, 0.1 mg/mL creatine phosphate sodium salt, 0.3 mg/mL adenosine 5’-triphosphate, 0.5 mg/mL creatine kinase, 0.12 mg/mL Na2SO4, 1 mg/mL nitrogenase isolated from azotobacter, and 0.1 atm C2H2. After water-bath heating at 30 °C for 6 h, 1 mL of NaCl saturated solution was added into the bottle to stop the reaction. Using C2H2 as the substrate, the ratio of C2H4/C2H2 of different samples could be used to evaluate the nitrogenase activity by gas chromatography.26 All experiments were performed at least three times to gain accurate data. 2.7. Circular Dichroism (CD) Spectroscopy of Nitrogenase and Nitrogenase/CDs. CD spectra of free nitrogenase and nitrogenase/CDs hybrids were tested with a Model 410 circular dichroism spectrometer with 50 nm/min scan speed and 1 mm of quartz cuvette. All CD spectra were corrected with the Tris−HCl buffer solution as the blank. To gain the accurate data, the CD spectra were tested at least three times. 2.8. Transmission Electron Microscopy (TEM) of Azotobacter. Azotobacter were grown on the medium with CDs (4 μg/ mL). After incubation for 72 h, azotobacter were collected and the cells were purified by centrifugation. Next, these bacteria were soaked in 5% glutaraldehyde to fix cells. After a series of dehydration processes, these bacteria were placed into the embedding agent. Ultrathin cross sections of these bacteria were observed by an FEI/ Philips Tecnai G2 F20 TWIN transmission electron microscope at 200 kV. 2.9. Cell Viability Assay. The cytotoxicity of CDs was determined in HeLa cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) using a microplate reader (Bio-Rad 680). Before CDs treatment, the HeLa cells were incubated in a 96-well plate at 37 °C in 5% CO2 for 24 h. Different concentrations (0−0.8 mg/mL) of CDs were added into the cells. The cells incubated without CDs were taken as reference. The cell viability was reflected by the absorbance at 570

3. RESULTS AND DISCUSSION Figure 1a shows the TEM image of CDs, revealing that CDs possess a monodisperse and uniform sphere morphology and the diameters of CDs are approximately 5 nm. The inset of Figure 1a exhibits the HRTEM image of CDs, indicating that the crystal lattice spacing of CDs is 0.21 nm, which belongs to the (110) lattice plane of graphitic carbon and implies the presence of a graphite structure.23,27 Figure 1b displays that the size distribution of CDs ranges from 3 to 7 nm, corresponding to the TEM image of CDs. Figure S1 shows the AFM image of CDs, which displays that the height of CDs is about 1.1 nm. As shown in Figure 1c, the FTIR spectroscopy of CDs (red line) indicates that there are different functional groups in the surface of CDs. A strong peak located at 3442 cm−1 corresponds to the vibration of hydroxyl bonds (O−H), and a narrow peak at 1635 cm−1 belongs to the vibration of carboxyl bonds (CO). The good water solubility of CDs is due to the presence of hydrophilic functional groups.27 Figure 1c (black line) is the UV−vis absorption spectrum of CDs. The typical absorption peak at 235 nm represents the π−π* transitions of CC bonds, which is consistent with the result reported before.27 As shown in Figure S2, the Raman spectrum (λex = 633 nm) of CDs was used to prove the disorder extent of the carbonaceous structure. The peaks at 1340 and 1600 cm−1 correspond to the D band and the G band, respectively. Figure 1d is the photoluminescence (PL) spectra of CDs under different excitation wavelengths, revealing that CDs have a strong fluorescence emission peak at 450 nm when excited at 330 nm. To investigate the fluorescence stability of CDs, the PL intensity of CDs at different temperatures, pH, and ion strengths were measured. As shown in Figure S3, the fluorescence intensity of CDs are stable at different temperatures, pH, ion strengths, and over time. To further study the composition and structure on the surfaces of CDs, the XPS spectra of CDs were determined. As shown in Figure 1e, the full survey spectrum shows that the C 1s peak is at 285 eV and the O 1s peak is at 532 eV. Figure 1f is the C 1s spectrum of CDs, which can be fitted into three peaks, corresponding to C−C bonds at 284.7 eV, C−O bonds at 286.2 eV, and CO bonds at 288.9 eV.29 The O 1s spectrum exhibited in Figure S4 can be fitted into two peaks at 531.7 and 532.8 eV, representing C−O and CO bonds, respectively.30 These results reveal that the carbonaceous structure and C

DOI: 10.1021/acsami.8b03758 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces oxygen-rich functional groups exist in the surface of CDs, which correspond to the FTIR spectrum of CDs. Figure S5a and S5b show the fluorescence decay of CDs, which are quenched by electron acceptor, 4-dinitrotoluene, and electron donor N,N-diethyl aniline. As shown in the insets of Figure S5a and S5b, the Stern−Volmer quenching constants (KSV = τF°kq) are 19 and 17 M−1, respectively. These experimental data reveal that CDs have a good ability of electron donating and accepting.27 The cytotoxicity of CDs was determined by MTT assay.28 Figure S6 is the cell viability of the Hela cells incubated with different concentrations of CDs. The Hela cells exposed to CDs at high concentration possess high cell viability, indicating that CDs have a good biocompatibility, which can be applied in biological field. Azotobacter are capable of converting N2 to NH3 by the catalysis of nitrogenase, which can be an excellent candidate for the study of biological nitrogen fixation.3,22 To investigate the effect of CDs on the N2 fixation activity of azotobacter, we cultured azotobacter on the medium supplemented with different concentrations of CDs (2, 4, 6, 8, 10, and 12 μg/ mL, experiment group) as well as the medium without CDs (control group). The N2 fixation activity of azotobacter was determined by ARA. The ratio of C2H4/C2H2 is used to speculate the N2 fixation activity of azotobacter. As shown in Figure 2a, the N2 fixation activity of azotobacter exposed to CDs (4 μg/mL) is increased 158% compared to that of the control, revealing that the addition of CDs significantly enhances the N2 fixation activity of azotobacter. Meanwhile, the nitrogen fixation activity of azotobacter treated with CDs at higher concentrations is reduced compared to that of the maximum. Moreover, to further verify the impact of CDs on the N2 fixation activity of azotobacter, the content of NH4+−N of azotobacter exposed to different concentrations of CDs was determined. As shown in Figure 2b, the content of NH4+−N of azotobacter treated with CDs (4 μg/mL) is increased by 37% compared to the control. The addition of CDs can promote the generation of ammonia, which corresponds to the results obtained by ARA. All of these results indicate that CDs can improve the nitrogen fixation of azotobacter. In the following study, we found that CDs could improve the growth of azotobacter. The growth measurement of azotobacter is based on the viable plating counting method and optical density at 600 nm (OD600).31 Figures 3a,3b and S7 are the photographs showing colonies of azotobacter cultured on the solid medium with or without CDs after 48 h. It can be observed that CDs have low toxicity to the azotobacter; meanwhile, CDs treatment has no obvious effect on the morphology of azotobacter. As shown in Figure 3c, the colonyforming unit (CFU) of azotobacter treated with CDs (4 μg/ mL) is higher than that of the control one. Moreover, the growth curve of azotobacter was determined by the optical density method. The azotobacter cells were incubated in liquid medium containing CDs of different concentrations for 48 h. As shown in Figure 3d, CDs have a dose-dependent impact on the growth of azotobacter, and CDs solution of 4 μg/mL is the optimal concentration for the growth of azotobacter, which is consistent with the results of the plating counting method. For the treatment of CDs at 4 μg/mL, the OD600 values were measured after incubation for 12, 24, 48, and 72 h. As shown in Figure 3e, the results clearly reveal that the addition of CDs at optimal concentration shows a positive effect on the growth of azotobacter at the initial stage.

Figure 3. (a, b) Photographs of azotobacter incubated on the medium without CDs and with CDs (4 μg/mL) showing colonies on plates after 48 h. (c) Normalized colony forming unit (CFU) of azotobacter treated with different concentrations of CDs. (d) Growth condition of azotobacter in liquid medium after 48 h incubation with CDs (2, 4, 14, and 20 μg/mL) in terms of measuring the optical density at 600 nm (OD600). (e) Growth curve of azotobacter treated with CDs (4 μg/ mL) after incubation of 12, 24, 48, and 72 h. All of the experiments were repeated three times at least.

The interaction between CDs and azotobacter was investigated with transmission electron microscopy (TEM), which is an effective means to reveal the ultrastructure of azotobacter and demonstrate the cellular uptake of CDs inside the cells. Figure 4a is the low-resolution TEM image of azotobacter exposed to CDs. As shown in Figure 4b, the presence of CDs is clearly observed in the azotobacter. Under high-magnification view of azotobacter, CDs can be detected in the cell wall and cytoplasm, indicating that CDs can enter the cytoplasm. Moreover, the HRTEM of azotobacter (Figure 4c) also exhibits that the CDs with a crystal lattice spacing of 0.21 nm can be observed in azotobacter. The results show that CDs could be absorbed by azotobacter, get into the cytoplasm, and have no negative effect on the entire morphology of the azotobacter. In the following experiments, to explore the mechanism in the process of biological nitrogen fixation, we first studied whether CDs could influence the nitrogenase activity. Nitrogenase is one of the key enzymes in the metabolism of nitrogen-fixing organisms, closely related to the process of nitrogen fixation. The determination of nitrogenase activity was performed by acetylene reduction assay. As shown in Figure 5a, the N2 fixation activity of nitrogenase/CDs hybrids is increased by approximately 56% compared to that of free nitrogenase, indicating that the treatment of CDs could improve the nitrogenase activity to some extent. It is possible that CDs may activate the nitrogenase and then accelerate the nitrogen fixation. D

DOI: 10.1021/acsami.8b03758 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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helix content of nitrogenase/CDs hybrids is increased compared to that of the free nitrogenase. To explain the interaction between CDs and nitrogenase, a series of experiments were performed. Figure 5c is the PL spectrum of CDs and CDs-Fe, which show that the fluorescence intensity of CDs-Fe is lower than that of CDs. Photoluminescence decay displayed in Figure S8 also indicates that Fe can be combined with CDs to form a CDs-Fe complex. As shown in Figure 5d, the fluorescence intensity of nitrogenase/CDs is lower than that of free nitrogenase, revealing that CDs can be combined with nitrogenase. These results show that CDs may improve the nitrogenase activity by combining with the nitrogenase and affecting the structure of enzyme. To further investigate the interaction between CDs and nitrogenase in nitrogenase/CDs, the CDs and nitrogenase were mixed in phosphate-buffered saline solution (pH = 7) and the nitrogenase/CDs hybrids were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). As shown in the inset of Figure 6a, the nitrogenase and nitrogenase/CDs stain the SDS-PAGE gels. The polyacrylamide gel electrophoresis results reveal that nitrogenase is much faster than nitrogenase/CDs hybrids, which prove that CDs and nitrogenase formed stable construction.28As is known, in azotobacter chroococcum, nitrogenase is the enzyme that is responsible for the reduction of N2 to NH3, consisting of two proteins: Fe protein and MoFe protein. In these two proteins, the F1 active site (Fe-S cluster in the Fe protein), functioning as the electron donor, can provide electrons to the F2 active site (MoFe cofactor in MoFe protein) where the reduction of nitrogen takes place.1,2,26 Besides, the size of nitrogenase is about 11.5 nm × 12.9 nm × 26.5 nm, which is much larger than the diameter of CDs (4.0 ± 0.5 nm). On the basis of the above results and analyses, we proposed a mechanism for the catalytic behavior of nitrogenase/CDs hybrids shown as Figure 6b. Because of the noncovalent interactions between CDs and

Figure 4. (a−c) TEM images of azotobacter. (d) Schematic diagram of azotobacter.

The catalytic activity of enzymes is highly related to the secondary structure of proteins. CD spectrum is one of the most powerful techniques for studying the secondary structure of proteins. To evaluate whether CDs could affect the nitrogenase structure, the CD spectrum of nitrogenase was determined by circular dichroism spectroscopy. Figure 5b is the CD spectra of nitrogenase (blue line) and nitrogenase/CDs hybrids (red line). For nitrogenase/CDs hybrids, the negative double peaks at 208 and 222 nm (α-helix) clearly decrease compared to that for free nitrogenase, indicating that the α-

Figure 5. (a) Nitrogenase activity of nitrogenase and nitrogenase/CDs separated from azotobacter. (b) Circular dichroism spectra of nitrogenase and nitrogenase/CDs. (c) PL spectra of CDs and CDs-Fe. (d) PL spectra of CDs and CD-nitrogenase (λex = 340 nm). E

DOI: 10.1021/acsami.8b03758 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 6. (a) SDS-PAGE of the free nitrogenase and nitrogenase/CD hybrids. (b) Schematic diagram of electron transfer pathways for nitrogenaseCD hybrids.

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nitrogenase, CDs are more likely to connect with nitrogenase to form nitrogenase/CDs hybrids. CDs have electron-donating and electron-accepting properties. CDs are easy to combine with Fe in nitrogenase (including F1 site and F2 site) by noncovalence. The insertion of CDs in nitrogenase led to the deepening of the α-helix structure in nitrogenase, which may help the F1 site to transfer more electrons to the F2 site. The above experiments suggest that for nitrogenase/CD hybrids, the F1 site transfers electrons to CDs and then CDs pass electrons to the F2 site of nitrogenase. Finally, electrons are used for the reduction of nitrogen.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Y.L.). *E-mail: [email protected] (Z.K.). ORCID

Hui Huang: 0000-0002-9053-9426 Mingwang Shao: 0000-0002-4220-463X Zhenhui Kang: 0000-0001-6989-5840 Author Contributions §

H.W. and H.L. contributed equally to this work.

Notes

The authors declare no competing financial interest.

4. CONCLUSIONS

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We have investigated the effect of CDs on the nitrogen fixation activity of azotobacter chroococcum and the catalytic behavior of nitrogenase. We found that CDs have a positive impact on the nitrogen-fixing activity of azotobacter and the activity of azotobacter treated with CDs (4 μg/mL) was increased by 158% than the control. The increase of nitrogen-fixing activity is because of the positive impact of CDs on bacteria growth and nitrogenase. The activity of nitrogenase/CDs was higher than that of free nitrogenase (increased by 56%). This is because CDs may combine with the nitrogenase, and spectroscopy analysis also proved that the addition of CDs could change the structure of nitrogenase, especially the α-helix. Nitrogenase contains the two proteins (Fe protein and MoFe protein), and CDs could combine with Fe in nitrogenase. It is possible that CDs with electron-donating and electron-accepting properties may accelerate the electron transfer among the active sites in nitrogenase, which improves the catalytic behavior of enzymes. This study shows that CDs are likely to be a novel candidate in the field of nitrogen fixation and these methods can be applied in other areas of microbiology and enzyme.

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ACKNOWLEDGMENTS This work is supported by the Collaborative Innovation Center of Suzhou Nano Science and Technology, the National Natural Science Foundation of China (51725204, 51572179, 21471106, 21771132, and 21501126), the China Postdoctoral Science Foundation (2017 M611902), the Natural Science Foundation of Jiangsu Province (BK20161216) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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REFERENCES

(1) Hoffman, B. M.; Lukoyanov, D.; Yang, Z. Y.; Dean, D. R.; Seefeldt, L. C. Mechanism of Nitrogen Fixation by Nitrogenase: The Next Stage. Chem. Rev. 2014, 114, 4041−4062. (2) Kim, J.; Rees, D. C. Nitrogenase and Biological Nitrogen Fixation. Biochemistry 1994, 33, 389−397. (3) Yates, M. G.; Planque, K. Nitrogenase from Azotobacter Chroococcum. Eur. J. Biochem. 1975, 60, 467−476. (4) Bohlool, B. B.; Ladha, J. K.; Garrity, D. P.; George, T. Biological Nitrogen Fixation for Sustainable Agriculture: A Perspective. Plant Soil 1992, 141, 1−11. (5) Li, H.; Kang, Z.; Liu, Y.; Lee, S. T. Carbon Nanodots: Synthesis, Properties and Applications. J. Mater. Chem. 2012, 22, 24230−24253. (6) Fan, Z.; Li, S.; Yuan, F.; Fan, L. Fluorescent Graphene Quantum Dots for Biosensing and Bioimaging. RSC Adv. 2015, 5, 19773−19789. (7) Zhu, S.; Meng, Q.; Wang, L.; Zhang, J.; Song, Y.; Jin, H.; Zhang, K.; Sun, H.; Wang, H.; Yang, B. Highly Photoluminescent Carbon Dots for Multicolor Patterning, Sensors, and Bioimaging. Angew. Chem., Int. Ed. 2013, 52, 3953−3957. (8) Zheng, M.; Liu, S.; Li, J.; Qu, D.; Zhao, H.; Guan, X.; Hu, X.; Xie, Z.; Jing, X.; Sun, Z. Integrating Oxaliplatin with Highly Luminescent Carbon Dots: An Unprecedented Theranostic Agent for Personalized Medicine. Adv. Mater. 2014, 26, 3554−3560.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b03758. AFM image; Raman spectrum; fluorescence stability; cell viability; O 1s spectrum; photoluminescence quenching spectra; photographs of azotobacter growth; photoluminescence decay of CDs and CDs-Fe (PDF) F

DOI: 10.1021/acsami.8b03758 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

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DOI: 10.1021/acsami.8b03758 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX