Green Fabrication of Carbon Dots upon ... - ACS Publications

Subscriber access provided by BUFFALO STATE

Article

Green Fabrication of Carbon Dots upon Photoirradiation and Their Application in Cell Imaging Qin Dai, He Zhao, Hongbin Cao, Yiqiu Wu, Zhi H.I. Sun, Xiaofeng Meng, Tianyu Wang, Guangfei Yu, Jingyi Lin, and Rong Hou ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.9b00305 • Publication Date (Web): 25 Apr 2019 Downloaded from http://pubs.acs.org on April 26, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 22 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

ACS Applied Nano Materials

Green Fabrication of Carbon Dots upon Photoirradiation and Their Application in Cell Imaging

Qin Dai, a,b He Zhao, a,* Hongbin Cao, a Yiqiu Wu,b Zhi Sun, a Xiaofeng Meng,c Tianyu Wang,d and Guangfei Yua,b, Jingyi Lin, a,b Rong Houe a

Beijing Engineering Research Center of Process Pollution Control, National Key Laboratory of Biochemical

Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China *Email: [email protected] b

University of Chinese Academy of Sciences, Beijing 100049, China

c

China University of Mining & Technology, Beijing 100083, China

d

Beijing Key Laboratory for Science and Application of Functional Molecular and Crystalline Materials,

Department of Chemistry, University of Science and Technology Beijing, Beijing 100083, China e

Changsha University of Science & Technology, Changsha 410114, China

ABSTRACT: The green and low-cost preparation of carbon dots (CDs) with excellent fluorescence plays a key role in cell-imaging applications. Herein, we develop a green photochemical approach for fabricating CDs. The produced CDs can be used directly in lung cancer A549 cell imaging without any tedious post-treatments. As nontoxic and green alternatives to prevent pollution, fatty acids in sole aqueous media are the only reactants used to synthesize CDs. Furthermore, the green process of photoirradiation avoids harsh conditions, such as strong acids and bases, strong oxidants, and high temperatures and pressures. In addition, the by-products remaining in the aqueous environment are fatty acid dimers in the form of vesicles, which can also be recycled as gemini surfactant resources. We demonstrate that this green synthetic method for fabricating CDs is suitable for fatty acids with different chain lengths, and therefore, exhibits considerable universality. Fatty acids, as readily available green resources in nature, and the green and simple synthesis method without additional pollution provides a new approach and perspective for the green synthesis of CDs for bioimaging. KEYWORDS: cell imaging, fluorescent carbon dots, photoirradiation, fatty acids, green synthesis INTRODUCTION Carbon dots (CDs) have emerged as attractive nanomaterials for cell-imaging applications in the past decades because of their outstanding tunable fluorescence (FL) properties, high chemical stability, low toxicity, excellent

1 ACS Paragon Plus Environment

ACS Applied Nano Materials 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

Page 2 of 22

biocompatibility, and surface modification flexibility.1-5 Numerous methods, such as hydrothermal method, laser ablation, electrochemical oxidation, microwave irradiation, hot injection, and pyrolysis, have been designed to synthesize CDs.6,

7

Green and mild synthetic methods have gradually emerged to reduce cost and expand CD

applications, and they have achieved certain results.8 Xia et al. prepared highly fluorescent carbon polymer dots at room temperature using an ascorbic acid/diethylenetriamine mixture under magnetic stirring for 72 h.4 Li et al. fabricated CDs using a monosaccharide/disaccharide/sodium hydrate (NaOH) mixture at room temperature.5 Huang et al. synthesized CDs at 90 °C using L-ascorbic acid and (N-(2-aminoethyl)-3-aminopropyl) tris-(2-ethoxy) silane as catalysts, which acted as stabilizing and passivation agents, respectively.6 Gong et al. used pumpkin with phosphoric acid (H3PO4) to obtain CDs at 90 °C.7 However, most of these methods still involve toxic reactants, more than one precursor, organic solvents, and additional additives, which not only require complicated separation processes after reaction but also potentially cause environmental issues. Thus, exploring a green and simple system using an environment-friendly precursor in aqueous phase is highly desirable for fabricating CDs. Fatty acids, as the bases of organisms, are widely present in plants, animals, and edible oils.9, 10 Thus, fatty acids, as precursors of CDs with the advantages of low cost, low toxicity, and high compatibility with organisms, can benefit applications in biochemistry or biodiagnostics.11-14 Meanwhile, sunlight is a natural sustainable green energy source, which provides a promising method for achieving the green preparation of functionalized materials.15, 16 Our previous studies indicated that photoirradiation can enhance the formation of CDs in methanol media.12 However, the replacement of the toxic solvent and the separation of products and by-products should be comprehensively considered and further investigated to develop a green alternative approach to CD fabrication. Here, we report a green photochemical method for fabricating CDs in aqueous media for the first time. The produced CDs can be collected directly for use in bioimaging. The synthetic method exhibits many distinct advantages over previously reported green CDs.8 First, our CDs have greener features and are closer to the definition of true green CDs. We demonstrate that not only the fatty acid precursor is green, but also the preparation process of light usage. Moreover, post-treatment is green. Second, our preparation conditions do not require high temperature and pressure and strong acid and alkali but can still obtain a higher absolute FL quantum yield (AFQY) of up to 2.8% in this mild process, which is comparable with that of CDs derived under harsh conditions. Third, the by-products can be recycled and reused without causing secondary pollution to the environment, and the method exhibits considerable universality, which is not always demonstrated by other approaches. Therefore, the proposed synthetic method is truly green, environment-friendly, direct, and does not produce secondary pollution.


Page 3 of 22 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


Accordingly, it conforms highly to the principles of green chemistry and provides a new perspective for the green fabrication of carbon nanomaterials. RESULTS AND DISCUSSION Green synthesis process: CDs were fabricated as shown in Figure 1. As a model, n-nonanoic acid (NA) was first dispersed in water (2%, v/v) and then exposed to photoirradiation with stirring. As reaction time extends, the appearance of new white emulsion indicates vesicle formation (Figure S1), according to previous reports.17 (Additional details regarding vesicles can be found in Figures S1–S5 and Scheme S1 in the ESI). The formed CDs with excellent FL were found to be wrapped inside vesicles through FL microscope (Figure S2), thereby indicating that vesicles acted as nanoreactors, similar to those in our previous study.12 Interestingly, the wrapped CDs can be released from nanoreactors and spontaneously accumulated on the water surface after standing for several days due to their hydrophobicity and lower density than water. A typical yellow color and strong blue FL under a 365 nm UV lamp demonstrated the existence of CDs.18, 19 Highly concentrated CDs undergoself-quenching (Figure S1b), which can be ascribed to excessive resonance energy transfer or direct π–π interaction.20 This characteristic is consistent with that of other reported CDs.20, 21 Although the original CDs primarily contained NA, the precursor type was not evidently fluorescent and was demonstrated to be nontoxic to cells at appropriate concentrations (Figure S17). This characteristic allows the CD solution to be collected and directly applied to bioimaging without any post-treatment. This result has not been successfully achieved in other reports, wherein tedious dialysis and separation are always required prior to application.18, 22-24 The NA–NA gemini surfactants that constituted the vesicles, which were demonstrated via mass spectroscopy (MS), further enhanced surface activity compared with NA monomer.25 These surfactants can be recycled for potential applications to enhanced oil recovery, contaminated soil remediation, and other fields.26, 27 Thus, no secondary pollution was produced in the entire reaction. The intermediates, as a function of time, were analyzed via MS to determine the mechanism of CD formation. The results showed that vesicles were composed of NA–NA dimers (313.2320 m/z) (Figures S4 and S5) in relation to previous reports.12,

17

In particular, NA–NA dimers (313.2320 m/z) and their oxidized state (329.2473 m/z)

increased along with reaction time, which is consistent with the increased vesicle number observed by the naked eyes (Figure S1). Other intermediates were identified as unsaturated structures, including unsaturated ketones, aldehydes, and acids (Figures 2 and S6, and Table S1). Among these intermediates, unsaturated acids, including



Page 4 of 22

C7H12O2 (127.1195 m/z), C9H16O3 (171.1077 m/z), and C28H46O8 (509.45744 m/z), presented an increased tendency along with time. By contrast, C14H24O4 (255.2363 m/z) initially increased and then decreased, which suggested that more unsaturated structures were formed. MS verified CD formation by showing a series of polymer peaks (Figure S6).28 Gel permeation chromatography (GPC) indicated that the number–average molecular weight and weight– average molecular weight of CDs were 38333 and 91308, respectively (Figure S3d). A polydispersity of 2.383 exhibited a comparatively wider distribution compared with those of other approaches,29-33 which may be attributed to the different lateral diameters and height distributions of CDs, as discussed in Physical and chemical structure characterization section. Our aforementioned findings and previous studies17,

34, 35

implied that the possible

mechanism of CD formation in water solvent is similar to that in the previous mixture system shown in Figure 3. First, the concentrated NA at the air–water interface absorbed light and then became excited, followed by the generation of NA free radicals (NA•) through reaction with NA or the hydroxyl radical (•OH).35 •OH and NA• radicals were demonstrated via in–situ electron spin resonance (ESR) spectroscopy and MS, respectively (Additional information can be found in Figure S5). Second, NA–NA dimers were formed through the recombination of NA• radicals and then underwent self-assembly into vesicles; unsaturated ketones/aldehydes/acids were produced through homolytic cleavage, intermolecular Norrish II, and NA• disproportionation.36 Third, the NA–NA dimers self-assembled into ca. 385 nm vesicles (Figure S3C), which are larger than those of the previous mixture system because water is not conducive to vesicle dispersion. Simultaneously, the unsaturated ketones/aldehydes/acids, which acted as carbon sources, were encapsulated into the bilayer or water chamber of the vesicles and transformed into CDs via dehydrogenation, polymerization, and self-assembly in nano-limited space under illumination. During the reaction, the vesicles acted as nanoreactors and enhanced the reaction due to their nanoconfined effect and reduction.12 (Additional details can be found in the ESI). Physical and chemical structure characterization: Transmission electron microscopy (TEM) and atomic force microscopy (AFM) characterizations were performed to validate the directly collected CDs. As shown in Figure 4, typical sphere-like CDs with a narrow diameter distribution were observed. The average diameter of CDs exhibited a slight increase from 3.23 nm to 3.57 nm with increased illumination time. This result may be mainly due to the fact that the CDs are continuously reduced in the vesicular nanoreactor, resulting in an increase in the conjugated structure. Notably, the CDs were larger than those in the previously mixed system12 primarily due to the larger vesicles in water media. That is, the size of CDs can be controlled by changing the size of vesicles through the adjustment of system composition. In general, most CDs prepared using the bottom-up method are highly


Page 5 of 22 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


amorphous in nature.37, 38 By contrast, a well-resolved crystal lattice observed in the high-resolution TEM image confirmed the crystalline structure of our three CDs with a labeled interplanar distance of 0.21 nm, which is consistent with the (100) lattice spacing of graphene along the [001] direction.39, 40 The AFM image shows that these CDs were monodispersed and exhibited similar particle heights of ca. 3 nm (Figure S8). The observed smaller size from AFM than that from TEM implied that these CDs collapsed during the sample preparation process for AFM testing, which is similar to a previous report.38 Nuclear magnetic resonance (NMR) was performed to verify the unsaturated structure of the three CDs. Aromatic hydrogen signals within the range of 6.2–8.6 ppm were observed in the 1H NMR spectra (Figure 5a). Correspondingly, the 13C NMR spectra also demonstrated the presence of aromatic carbon within the range of 100– 135 ppm (Figure 5b).41 The spectra within 60–80 ppm were attributed to the C-O-C functional groups.19 These findings clearly demonstrated that the type of unsaturated carbon increased as reaction time was extended, which was attributed to the newly generated unsaturated structure of CDs. Fourier-transform infrared spectroscopy (FTIR) was performed to identify the functional groups of the three CDs (Figure 5c). The characteristic bands at 1712 cm−1 and 1562 cm−1 were ascribed to the C=O stretching vibration in the carboxyl group and the C=C stretching, respectively. An absorption band at 1118 cm−1 could be assigned to the symmetric stretching modes of C–O–C from either ether or epoxy. The peaks at 2854–2954 cm−1 and 1415–1458 cm−1 resulted from the vibration of C–H. Therefore, the surface functional groups on the CDs were predicted to be carboxyl (C=O) and epoxide/either (C–O–C) groups. No evident hydroxyl (–OH) signal was observed, which resulted in the hydrophobicity of CDs. The considerable increase in the ratio of C=C/C=O with reaction time also provided evidence that the conjugation degree was increased, which is in close agreement with the results obtained via NMR analysis. X-ray photoelectron spectroscopy (XPS) was performed to confirm the formation and content of the aforementioned functional groups in CDs. The results showed that the CDs were composed of C and O elements, and the atomic ratios of C1s of the three CDs were 68.87%, 73.51%, and 74.24% (Figure S9). The four peaks at 288.2, 285.3, 284.8, and 284.4 eV were ascribed to the O=C-O, C-O-C, C-C, and C=C groups, respectively (Figure 5d). The C=C contents of CDs with increasing reaction time changed from 23.09% and 24.70% to 26.71%, thereby indicating that the conjugation degree of the structures increased (Figure 5e). The decreased O=C-O content suggested that a considerable amount of NA was decarboxylated and then transformed to CDs, as indicated by the FTIR and NMR results.



Page 6 of 22

Optical properties: The FL properties of CDs are important for their applications in bioimaging and biosensors. Therefore, the synthesized CDs were diluted in ethanol for the subsequent optical characterizations. The ultraviolet– visible (UV–Vis) spectra (Figure 6a) indicated that the strong absorption peaks in the region of 235–246 nm were ascribed to the π–π* transition of the CD core, and the weak shoulder peaks at 272–302 nm were related to the n– π* transition of the carbonyl group on the surface of particles.42 With the extension of irradiation time, the π–π*, n– π*, and visible peaks (from 400 nm to 600 nm) redshifted due to increased unsaturated C=C and decreased O=C contents.43 The color of CDs varied from light to dark yellow under room light as photoreaction time increased (insets in Figure 6a). This behavior is consistent with the generation of additional conjugated structures.44 Under excitation at 365 nm, the emission colors of CDs were blue FL (insets in Figure 6a), which originated from the carbon core and the surface state of CDs.45 Their maximum emission peaks exhibited a slight redshift that changed from 449 nm to 453 nm and 459 nm as reaction time was prolonged (Figure 6b), which resulted from an increase in conjugation degree. Correspondingly, their maximum excitations were 367, 365, and 370 nm (Figure 6c). In accordance with the excitation and emission spectra, the stokes of the three CDs were calculated to be 82, 88, and 89 nm, thereby showing an increasing trend (Table S1). These values indicated that the self-absorption of CDs was minimal,46 which is beneficial for efficient FL emission.47 In the 3D fluorescent spectra (Figure 6d), the FL of CDs could be excited within a wide range of wavelengths, and the fluorescent centers of CD solutions with different reaction times were located at ca. 450 nm. Subtle changes in the shape of the FL center could be attributed to changes in the core and surface structure of the CDs with prolonged reaction time. The emission presented pronounced excitation-dependent properties, which are typical optical characteristics of CDs and consistent with another work.41 This phenomenon originates from the red-edge effect, i.e., if the solvation dynamics is not an order of magnitude faster than the FL lifetime, then the fluorophore can emit simultaneously while the excited state’s energy is being reduced, thereby creating a time-dependent emission energy.43 With the extension of photoirradiation time from 4, 12 to 16 h, the FL lifetimes of CDs were 2.43, 2.52, and 2.04 ns, respectively, under an excitation of 370 nm and their maximum emission (Figure S11). Moreover, FL lifetime exhibited emission wavelength-dependent properties under an excitation of 370 nm (Figure S12), which were predominantly caused by the different size of small sp2 carbon clusters which is also called polydispersity in CDs.

48

It implies that CDs with different sizes have different band gaps and can be excited by

light of different wavelengths, thereby, exhibiting different FL lifetimes under excitation of light of different


Page 7 of 22 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


wavelengths.48 Similarly, different FL lifetime can be obtained by detecting different wavelength emission under the same excitation. The AFQY of CDs tended to increase with photoirradiation time. The AFQYs of CDs prepared for 4, 12, and 16 h were 2.27%, 2.43%, and 2.80%, respectively (Figure S13), which are sufficient for cell imaging (1%–2% AFQY is required). The increases in AFQY could be attributed to the enhanced conjugation degree of CDs.49 The maximum AFQY and FL lifetime of the CDs in this study are comparable with and even superior to those of previously reported CDs produced using harsh methods.50 Photostability is also crucial for CD application given that CDs are synthesized via photoirradiation. Figure S16 shows that our fabricated CDs exhibited superior photostability even under 12 h of irradiation with a 365 nm UV lamp in both NA and ethanol solution. The results further confirmed that the CDs were generated only in the presence of vesicles and the air–water interface. UV irradiation exerted no effect on the formation of CDs when CDs were dissolved in total organic solvent. Moreover, CDs were well solubilized and suspended in PBS and whole serum without any aggregation and any changes in FL intensity after being kept at room temperature for several days. Similarly, saturated fatty acids, including hexanoic acid (C6), heptanoic acid (C7), and octanoic acid (C8), were used as precursors. After 5 h of illumination, the obtained CD solutions emitted generally blue FL under UV lamp irradiation. And when changing from a low concentration to a high concentration, these blue FL intensity of CDs solutions was decreased obviously due to the fact that the high concentration of CDs causes the CDs to aggregate more easily. That is generally called conventional aggregation-caused quenching (ACQ) (Figure S14).51 The 3D fluorescent spectra also show that their FL centers were located at approximately 450 nm, presenting typical excitation-dependent properties (Figure S15). These results demonstrated that the green synthetic route of CDs exhibits high universality. Cytotoxicity and cell-imaging application: Low toxicity and considerable biocompatibility are generally prominent characteristics of saturated fatty acids;52 however, the cytotoxicity of the corresponding as-synthesized CDs is unknown. Therefore, in vitro MTT assay was conducted using A549 cells to verify the cytotoxicity of the original NA and the obtained CDs without any post-treatment. Figure S17 shows that the A549 cells treated with the original NA and CDs retained over 100% viability at extensive concentrations ranging from 15 μM to 3840 μM. Therefore, the cells exhibited higher tolerance concentration for NA than that in the previous report.53 These results indicate that pure NA and the CD solution exhibit excellent properties of low toxicity and biocompatibility. The



Page 8 of 22

possible reason for cell viability higher than 100% was that a higher amount of fatty acids was adsorbed or decorated on the CDs with increasing CD concentration. The biocompatible fatty acids acted as the carbon sources of cells, which promoted cell growth. After low cytotoxicity and good biocompatibility were demonstrated, bioimaging experiments were monitored using an FL microscope, as shown in Figure 7. Prior to the addition of CDs, no FL signal was observed on the A549 cells. While being cultured for 3 h with the CD solutions at a concentration of 250 μM, both cell membrane (marked

with red arrows) and cytoplasm (marked with yellow arrows) emitted clear blue and green FL and weak red FL under excitations of 408, 488, and 561 nm, respectively. In contrast, no obvious FL was observed in the nuclear

position (marked with white circles). The observation of the bright blue and green area inside the cells indicated the translocation of CDs through the cell membrane. In addition, they showed no blinking and low photobleaching, because FL intensity of cell showed no obvious changes under the confocal laser for nearly 30 minutes. The aforementioned results hint the prepared CDs’ good photostability and considerable potential as bioimaging materials.

CONCLUSIONS A facile, environment–friendly, and effective one-pot photochemical fabrication method for CDs is reported in this paper. The produced CDs, which demonstrated hydrophobicity and outstanding properties, were collected directly from the water surface and successfully applied to cell imaging without any post-treatment. This paper is the first to report the green synthesis of CDs in water media through the photoirradiation of fatty acids. The method achieved considerable universality for fatty acids with different carbon chain lengths (C6–C9). It provides one of the most environment-friendly methods for the synthesis of CDs with low toxicity and biocompatibility and facilitates the application of CDs to cell imaging or other related biological fields.

EXPERIMENTAL METHODS Chemicals NA (≥ 97.5%) was provided by Sinopharm Chemical Reagent Co., Ltd. Hexanoic acid (AR, 99.0%), heptanoic acid (AR, 98.0%) and n-Octanoic acid (AR, 99%) were purchased from Aladdin. Ethanol and acetonitrile used in this study were of Optima LC/MS grade and were available from Fisher Scientific (Fair Lawn, NJ). Deuterated


Page 9 of 22 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


methanol was provided by Cambridge Isotope Laboratories, Inc.. Petroleum ether was obtained from Beijing Chemical Works and acetic acid was acquired from Xilong Chemical Co., Ltd. Dimethyl sulfoxide (DMSO) and ethyl acetate were purchased from Xilong Scientific Co., Ltd. 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) and 3(4,5-Dime-thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Dulbecco's Modified Eagle Medium (DMEM), penicillin (10,000 U/mL), streptomycin (10 mg/mL), and fetal bovine serum (FBS) were obtained from Thermo. Paraformaldehyde (1% PFA) and phosphate buffered saline (PBS, pH = 7.4) were provided by Shanghai Yu Bo Biotechnology Co., Ltd. All other reagents used were obtained commercially at analytical grade without further purification. Lung cancer A549 cells were purchased from Tumor Cell Bank of Chinese Academy of Medical Sciences. 12 Synthesis NA (2%, v/v) was added to the water in cylindrical quartz tubes. No photo-initiators or catalysts were added throughout the entire photoirradiation process. After ultrasonic treatment and even stirring, the quartz tubes were placed in a photo-reactor equipped with a 500 W high-pressure mercury lamp (λ > 254 nm, light intensity = 12 mW/cm2, shown as the light spectrum in Figure S7) with magnetic stirring. Acids with different carbon chains underwent identical photoreaction conditions as that of NA. The reactions were not deoxygenated and the photoreactor was maintained at ambient temperature using a low-temperature water pump (ZL-500). Reactions were stopped at predetermined time points. After being placed for a few days, the yellow liquid accumulated on the liquid surface (Figure 1 and S3b) and can be directly collected for the cell imaging application. For various physical and chemical characterizations, CDs were separated and purified using thin layer chromatography silica gel plate and separated by 0.22 µm microporous membrane to remove NA and other impurities. Petroleum ether, ethyl acetate, and acetic acid were applied as eluents (v/v/v = 18:3:3). Finally, purified CDs with different photoirradiation times and different carbon chains were obtained after being subjected to vacuum drying. Residual white emulsion was collected directly for its component identification.12 Physical and chemical Characterizations Selected yellow liquids and collected vesicles by centrifugation were detected by electron spray ionization time-offlight mass spectrometry (ESI-TOF-MS) under negative mode, respectively. The mass analyzer was run at a scanning mode from m/z 100 to 1000. Nitrogen was used as desolvation gas and maintained at a flow rate of 10 L /min. The desolvation temperature was set at 350 °C. The reaction products were verified through the following



Page 10 of 22

strategy in accordance with MS analysis: The molecular formulas of each species present in the reaction mixtures but not in control samples were derived from the accurately measured mass and isotope patterns. The injection volume of the sample was 5 µL. The adduct of NA• radical and 5-Dimethyl-1-Pyrroline-N-Oxide (DMPO) was detected by ESI-TOF MS under positive mode. Hydroxyl radicals originated from NA aqueous solution after 1 minute of photoirradiation were detected by an in-situ ESR (E 500, Bruker, Germany) equipped with a light source. And DMPO was used as a spin-trapping agent. The polydispersity and molecular weight of CDs were measured by GPC using 1515 GPC instrument with Waters styragel HR1, HR4 columns. Tetrahydrofuran used as the mobile phase and its flow rate was 1 mL/min. Linear poly(methyl methacrylate) with narrow molecular weight distribution were used as the standards to calibrate the apparatus. Dynamic Light Scattering (DLS) was used to detect the zeta potential and diameter of the vesicles. Vesicle solution was diluted by 1000-fold with water (viscosity 0.88cP, reflection index 1.330) and determined with 173 backscatters with water as the dispersed phase under ambient temperature. The samples were equilibrated for 120 seconds and tested for three times to obtain the average data. The corresponding pH of the sample was measured by a pH meter (PHSJ-6L) after DLS characterization. The morphology and structure of samples were investigated through high solution field-emission transmission electron microscopy HRTEM (JEM-2100F) at an accelerating voltage of 200 kV. Samples for HRTEM characterization were prepared by placing 4~6 drops of colloidal solution on a carbon-coated copper grid and dried at ambient temperature. The size of the samples was estimated by using Nanomeasurer 1.2 software. CDs height was obtained using an atomic force microscope (AFM, Multimode 8, Bruker, Germany) under tapping mode using tap-300G silicon cantilevers with a tip radius

Green Fabrication of Carbon Dots upon ... - ACS Publications

Recommend Documents