Oxygen-driven, high-efficiency production of nitrogen-doped carbon dots from alkanolamines and their application for two-photon cellular imaging

Abstract

A novel oxygen-driven method has been developed for low-cost, large-scale, and high-efficiency production of nitrogen-doped carbon dots (N-C-dots) by bubbling pure oxygen into monoethanolamine (MEA) under heating conditions. We find that the addition of pure oxygen significantly increases the reaction rate, and makes feasible one-pot gram scale fabrication (3.36 g) of highly photoluminescent N-C-dots in a couple of hours (2.0 h). With an instantaneous nucleation and gradual growth mechanism, precise control over the particle size of the N-C-dots from 2.0 to 16.1 nm is achieved by simply prolonging the heating time from 0.5 to 4.0 h. The as-prepared N-C-dots contain aromatic CN heterocycles in the core and have plentiful hydrophilic groups on the surface. Practically, the oxygen-driven method can be used to synthesize fluorescent N-C-dots from other alkanolamines such as diethanolamine (DEA) and triethanolamine (TEA), which shows general universality. Due to the strong up-conversion photoluminescence, good aqueous dispersibility, high photostability, excellent biocompatibility, and low cytotoxicity, the N-C-dots are demonstrated to be promising two-photon probes for high contrast bioimaging applications.

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Introduction

Carbon dots (C-dots), a new class of photoluminescent carbon nanomaterials, have generated high amounts of excitement due to their unique optical properties, low environmental hazard, excellent biocompatibility, and robust chemical inertness.^1–4 These superiorities make C-dots promising alternatives to heavy metal-based quantum dots for promising applications in bioimaging,^5–7 drug delivery,⁸ sensors,^9–12 and photocatalysis.^13,14 To date, a variety of synthetic strategies have been developed for synthesis of C-dots, which can be generally classified into top-down and bottom-up routes. Top-down routes rely on cutting large graphene-based materials into nanosized C-dots by arc discharge,¹⁵ laser ablation,¹⁶ plasma treatment,¹⁷ or electrochemical oxidation.¹⁸ Unfortunately, these routes require special equipment, harsh synthesis conditions, and complicated synthetic procedures, which hinder their scale-up process and limit practical applications. Bottom-up routes involve microwave/ultrasonic pyrolysis,^19,20 hydrothermal treatment,^21–26 acidic oxidation,^27,28 and thermal oxidation^29–32 of molecular precursors towards C-dots. Among them, the thermal oxidation method pioneered by Bourlinos et al. can be conducted in open air at mild temperature without any elaborate equipment, and has potential to be an effective and convenient approach for large-scale production of C-dots. Recently, Chou group employed this method to prepare C-dots by heating glycerol under 1 atmosphere air pressure;³³ Chen and co-workers also synthesized C-dots by thermal treatment of polyethylene glycol in air.³⁴ In both cases, the oxygen in air acted as an essential oxidant for C-dots formation. However, the volume fraction of oxygen in air is only 0.21, and the oxygen in air need to diffuse into glycerol or polyethylene glycol to contact with the precursor molecules for reaction, thus seriously limiting the reaction rate for fast production of C-dots. So far, it is still highly desired to explore a facile method for low-cost, large-scale, and high-efficiency production of fluorescent C-dots with uniform morphology and controllable size.

On the other hand, doping heteroatoms especially nitrogen into C-dots has been actively pursued because the introduction of nitrogen atoms into carbon frameworks with lone electron pairs can effectively tune the intrinsic properties of C-dots, such as electronic characteristics, surface and local chemical features.^35–37 For synthesis of nitrogen-doped C-dots (N-C-dots), apart from the synthetic method, the selection of suitable precursors is another key factor. Alkanolamines are chemical compounds that contain both hydroxy (–OH) and amino (–NH₂, –NHR, and –NR₂) functional groups. Simple alkanolamines such as monoethanolamine (MEA), diethanolamine (DEA) and triethanolamine (TEA) are cheap industrial feedstocks and widely used as solvents, synthetic intermediates, and high-boiling bases in organic chemistry. We assume that alkanolamines may serve as appropriate carbon and nitrogen sources for N-C-dots synthesis.

In the present paper, we demonstrate a novel oxygen-driven method to synthesize N-C-dots by bubbling pure oxygen into alkanolamines under heating condition. The synthetic procedure is illustrated in Fig. 1. This method is actually simple and economical, and provides an efficient route for scalable production of highly photoluminescent N-C-dots in one pot. MEA is a typical case. Initially, we found that thermal treatment of MEA in open air led to a pale yellow dispersion of N-C-dots. However, the reaction rate was rather slow. If pure oxygen was bubbled into MEA, the reaction rate significantly increased. For instance, in the oxygen flow rate of 100 mL min⁻¹, the production efficiency of N-C-dots was improved by nearly 50 times compared with the condition that no pure oxygen was added. It is noticeable that our method enables wide-range size tuning of N-C-dots from 2.0 to 16.1 nm by simply changing the heating time. The oxygen-driven method is also universal, as other alkanolamines including DEA and TEA are feasible as precursors for producing N-C-dots. Importantly, different from the previously reported one-photon bioimaging application of C-dots, the N-C-dots with strong up-conversion photoluminescence, good aqueous dispersibility, high photostability, and low cytotoxicity, have been successfully applied as excellent two-photon fluorescent probes for cellular imaging under near-infrared (NIR) excitation.

Fig. 1 A schematic illustration of oxygen-driven, high-efficiency production of nitrogen-doped carbon dots from alkanolamines.

Experimental

Reagents and materials

Monoethanolamine (MEA), diethanolamine (DEA), triethanolamine (TEA), ethylenediamine and ethylene glycol were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). 3-(4,5-Dime-thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The deionized water was produced through a Millipore water purification system (≥18 MΩ, Milli-Q, Millipore). All other reagents were of analytical grade and used without further purification.

Synthesis of N-C-dots

Different alkanolamines (MEA, DEA, TEA) and ethylenediamine were used for fabricating N-C-dots (C-dots are prepared from ethylene glycol). For a typical synthesis, 100 mL of MEA was gradually heated to 170 °C under reflux in air. When the temperature reached 170 °C, pure oxygen in a certain flow rate (0, 25, 50, and 100 mL min⁻¹) was bubbled into MEA. The color of the solution turned from colorless to yellow, orange, red and brown, indicating the formation and growth of the N-C-dots. The solution of the N-C-dots naturally cooled to room temperature. After evaporating the unreacted MEA that serves as the solvent by vacuum distillation and drying the sample under vacuum oven, solid N-C-dots were obtained.

Calculation of quantum yield

The quantum yield of the N-C-dots was measured using quinine sulfate as the standard and calculated with following equation:

QY_x = QY_std (I_x/I_std)(A_std/A_x)(η_x²/η_std²)

where the subscript “x” designates the N-C-dots, the subscript “std” designates quinine sulfate, “QY” stands for the quantum yield, “I” stands for the integrated PL intensity, “A” stands for the absorbance, and “η” stands for the refractive index of the solvent. Quinine sulfate (quantum yield: 54.6%) was dissolved in 0.1 M H₂SO₄ (refractive index: 1.33) and the N-C-dots were dissolved in water (refractive index: 1.33). In order to minimize reabsorption effects, absorbance value of the individual solution was kept below 0.10 at the excitation wavelength (360 nm).

Cytotoxicity against Hela cells

The cytotoxicity test of the as-synthesized N-C-dots was carried out using MTT assay on Hela cells according to the previously reported literature.²⁵ In detail, 100 μL suspension of Hela cells (5 × 10⁴ cells mL⁻¹) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin were added to per well of 96-well plates and incubated in a 5% CO₂ humidified incubator at 37 °C for 24 h. The N-C-dots were introduced into the wells in a concentration of 100, 200, 300, 400, 500, and 600 μg mL⁻¹ and incubated for 48 h. The medium was removed and cells were washed with phosphate-buffered saline (PBS). Then, 20 μL of 5 mg mL⁻¹ MTT solution was added to each cell well. The 96-well plates were further incubated for 4 h, followed by removing the culture medium with MTT, and then 200 μL of DMSO was added. The optical density of the mixtures at 490 nm was measured. Cell viability was expressed as percentage of absorbance relative to control. Experiments were performed in triplicates, with nine replicate wells for each sample and control per assay.

Two-photon bioimaging

An aqueous solution of N-C-dots (400 μg mL⁻¹) was passed through a 0.2 μm sterile filter membrane. The filtered fluorescent suspension (40–60 μL) was mixed with the culture medium (200 μL) and then added to the well of the confocal dish in which Hela cells were grown. After incubation for 1 h, the medium was removed and the cells were washed thoroughly three times with PBS and kept in PBS for two-photon bioimaging. Cellular uptake of the N-C-dots by Hela cells tracked via confocal microscopy (Leica TCS SP5) and the emission was measured over the range of 410–550 nm with a NIR excitation at 760 nm.

Instruments and measurements

Transmission electron microscopy (TEM) images were taken on a JEM-2100 operating at an accelerated voltage of 200 kV. X-ray diffraction (XRD) measurement was performed with a Shimadu XRD-6000 powder X-ray diffractometer with CuKα radiation (1.54056 Å). The Raman spectrum of as-prepared samples was recorded at ambient temperature on LabRAM ARAMIS Confocal Raman Microscope with a laser at an excitation wavelength of 532 nm. X-ray photoelectron spectroscopy (XPS) measurements were carried out with a PHI 5000 VersaProbe X-ray photoelectron spectrometer. Fourier transformed infrared (FTIR) spectrum was collected in the KBr medium on a Nicolet 6700 FTIR spectrometer. Elemental analysis was measured on a vario EL II elemental analyzer. ¹H NMR and ¹³C NMR spectra were carried out with a Bruker Avance III 400 spectrometer by dispersing 20 mg of N-C-dots in 500 μL of D₂O. Ultraviolet-visible (UV-vis) absorption spectra were recorded on a UV-3600 spectrophotometer. Photoluminescence (PL) spectra were obtained on a RF-5301 PC spectrophotometer. Time-resolved fluorescence spectrum and up-conversion PL spectrum were measured with an Edinburgh FLS 920 spectroscope. Two-photon cellular imaging was done under Leica TCS SP5 confocal microscope with a NIR excitation at 760 nm.

Results and discussion

Structure and composition of the N-C-dots

Thermal treatment of 100 mL of MEA in the oxygen flow rate of 50 mL min⁻¹ at 170 °C for 3.0 h resulted in a reddish brown dispersion of N-C-dots. The morphology and structure of the N-C-dots were investigated by TEM, XRD and Raman measurements. The TEM image (Fig. 2a) shows that the N-C-dots are nearly monodisperse nanoparticles and have a relatively narrow size distribution with an average diameter of 15.4 nm (Fig. 2b). The high resolution TEM (HRTEM) image (Fig. 2a, inset) reveals that the diffraction contrast of the N-C-dots is very low and without any well-resolved lattice fringes, indicating their amorphous nature. The XRD pattern (Fig. 2c) exhibits a broad peak at 22.26°. The calculated interlayer spacing is 0.399 nm, larger than that of natural graphite (0.335 nm), which is attributed to the presence of abundant functional groups surrounding the edges of graphene layers in the N-C-dots.³⁸ The Raman spectrum (Fig. 2d) presents a D peak (sp³) at 1358 cm⁻¹ and a G peak (sp²) at 1562 cm⁻¹, demonstrating partial disordered graphite-like structure of the N-C-dots.³⁵

Fig. 2 (a) TEM image, (b) size distribution, (c) XRD pattern, and (d) Raman spectrum of the N-C-dots. Inset in (a): HRTEM image of the N-C-dots.

XPS was performed to analyze the surface state and element composition of the N-C-dots. The survey spectrum (Fig. 3a) shows three typical peaks of C_1s, N_1s and O_1s with corresponding content of 68.56%, 13.29%, and 18.15%. The C_1s spectrum (Fig. 3b) exhibits five carbon signals at 284.0, 284.5, 286.0, 286.5 and 288.0 eV, attributed to C=C, C–C, C–N, C–O, and C=N/C=O groups respectively.²³ The N_1s spectrum (Fig. 3c) can be deconvoluted into three different nitrogen species, associated with graphitic N at 398.5 eV, pyridinic N at 399.2 eV, and pyrrolic N at 400.1 eV,³⁹ which indicates that nitrogen atoms have doped into the carbon framework of the nanodots and existed in the form of aromatic CN heterocycles. Two characteristic oxygen state of C=O at 531.7 eV and C–O at 532.9 eV are observed in the O_1s spectrum (Fig. S1†).²³ The elemental analysis reveals that the N-C-dots are composed of C 60.25 wt%, N 14.89 wt%, H 5.53 wt%, and O 19.33 wt% (calculated), agreeing well with the XPS investigation. The FTIR spectrum (Fig. 3d) displays the characteristic absorption bands of O–H/N–H stretching vibrations at 3354 cm⁻¹, C–H stretching vibrations at 2933 and 2879 cm⁻¹, C=O stretching vibration at 1655 cm⁻¹, C–N stretching vibration at 1441 cm⁻¹, C–H bending vibration at 1354 cm⁻¹, and C–O stretching vibration at 1061 cm⁻¹. The NMR studies distinguish sp²-hybridized carbons from sp³-hybridized carbons. The sp²-hybridized carbons are detected in 6–8 ppm in ¹H NMR spectrum (Fig. 3e) and 100–160 ppm in ¹³C NMR spectrum (Fig. 3f). The ¹³C NMR spectrum also reveals the presence of C=O carboxylic/carbonyl carbons in 160–180 ppm. All the above investigations indicate that the as-synthesized products are carbon-rich, nitrogen-doped nanodots, which contain aromatic CN units and are functionalized with abundant functional groups including hydroxyl, epoxy, carbonyl, and carboxyl.

Fig. 3 (a) XPS survey spectrum, (b) C_1s spectrum, (c) N_1s spectrum, (d) FTIR spectrum, (e) ¹H NMR spectrum, and (f) ¹³C NMR spectrum of the N-C-dots.

Optical properties of the N-C-dots

The aqueous dispersion of the N-C-dots is yellow in daylight (Fig. 4a, left inset). It exhibits a bright blue color with UV light irradiation (Fig. 4a, right inset). In the UV-vis absorption spectrum (Fig. 4a), an absorption peak centered at 287 nm is clearly observed, which is ascribed to the n–π* transition of C=O or N=O groups. The PL spectra (Fig. 4b) show the strongest emission peak at 464 nm with full width at half-maximum (FWHM) as narrow as 83 nm for excitation at 380 nm. The emission peak gradually red-shifts from 464 to 520 nm with intensity slowly decreasing when the excitation wavelength is tuned from 380 to 480 nm. The excitation wavelength dependent PL behavior of the N-C-dots results from the different particle sizes and the diverse surface emissive trap sites.¹⁶ Fig. 4c illustrates the PL decay profile of the N-C-dots, which can be well fitted into a double-exponential decay function with a goodness-of-fit χ² = 1.018. The two components of decay lifetime are τ₁ = 4.05 ns (54.05%) and τ₂ = 14.07 ns (45.95%), suggesting that two different emissive centers, namely electronic conjugate structures and surface traps, exist.⁴⁰ The average PL lifetime is 8.65 ns. The quantum yield of the N-C-dots is determined to be 12.3% by selecting quinine sulfate (54.6% in 0.1 M sulfuric acid) as a standard, higher than the values of most reported doping-free C-dots.^19,33,34,40 Noticeably, the N-C-dots present an efficient up-conversion PL feature with long-wavelength light excitation (Fig. 4d), which originates from two or multiphoton active process.⁵ Some previous studies demonstrated that the up-conversion PL of the C-dots was not a real signal, because it was derived from the second-order diffraction light of wavelength λ/2 which coexisted in the selected light (first-order) of wavelength λ.^41–43 In our experiment, we employed a 395 nm long-pass filter in the excitation channel (between the excitation source and the sample) to eliminate the light at 370 nm (740/2) using a 740 nm excitation wavelength, and found that an intensive fluorescence peak at around 470 nm could be detected (Fig. 4d), which confirmed the presence of the up-conversion PL of the N-C-dots. The up-conversion PL feature opens new opportunities for N-C-dots based two-photon bioimaging applications⁵ as well as high-performance photocatalysts design.¹³

Fig. 4 (a) UV-vis absorption spectrum, (b) PL spectra, (c) PL decay profile, and (d) up-conversion PL spectra of the N-C-dots. Insets in (a): photographs of the aqueous solution of the N-C-dots excited by daylight and a UV lamp (365 nm). Inset in (b): normalized PL spectra of the N-C-dots.

The PL intensity of the N-C-dots to the effects of pH, ionic strength, and UV exposure time were investigated. As shown in Fig. S2,† the PL intensity drops rapidly as pH >9, but keeps at a relatively high value in a solution of pH 2–8. There is no distinct decline of the PL intensity at different ionic strength (Fig. S3†). Moreover, no photobleaching is found in continuous UV excitation for hours (Fig. S4†). These results prove the excellent PL stability of the N-C-dots.

Effect of reaction time on synthesis of N-C-dots and a proposed formation mechanism

To gain insight into the formation process of the N-C-dots, time-dependent experiments were carried out. As shown in Fig. 5a, refluxing 100 mL of MEA in the oxygen flow rate of 50 mL min⁻¹ at 170 °C leads to a gradual color change that from colorless to yellow, orange, red and brown, which implies the formation and growth of the N-C-dots. The temporal evolution of UV-vis absorption spectra (Fig. 5b) reveals that, at the start of the reaction, there is no absorbance beyond 240 nm. When the reaction proceeds for 0.5 h, an absorption peak at 290 nm emerges, indicating the nucleation of the N-C-dots. With heating time, the absorption peak slightly blue-shifts and the absorbance steadily enhances, which is suggestive of the particle growth and the increasing concentration of the N-C-dots.³⁸ Corresponding to UV-vis absorption spectra, the PL monitoring (Fig. 5c) shows that, an emission peak arises when MEA is heated for 0.5 h, and the emission intensity increases as the reaction time is further prolonged. It is found that the quantum yield of the N-C-dots gradually goes up from 3.6 to 12.2% in the first 2.0 h, and then remains nearly unchanged (Fig. 5d).

Fig. 5 (a) Photographs of the N-C-dots prepared at different reaction time (from left to right: 0, 10, 30 min, 1.0, 1.5, 2.0, 3.0, and 4.0 h). The reaction temperature was 170 °C, and the oxygen flow rate was 50 mL min⁻¹. (b) UV-vis absorption spectra and (c) PL spectra of the N-C-dots prepared at different reaction time. The solutions of the N-C-dots for test were diluted 400 times. (d) Quantum yields of the N-C-dots prepared at different reaction time. Insets in (b) and (c): enlarged UV-vis absorption spectra and PL spectra of the N-C-dots prepared in 0, 10, and 30 min.

TEM images of the N-C-dots at different reaction stages are recorded (Fig. 6). Briefly speaking, the N-C-dots first nucleate and then grow up with size distribution being broader during the heating process. For a detailed description, the average diameter of the N-C-dots determined by TEM as a function of reaction time is presented (Fig. S5†). It illustrates that the nuclei of the N-C-dots with average diameter of 2.0 nm generate in the first 0.5 h. The particle size slightly increases from 2.0 to 2.6 nm by prolonging the heating time from 0.5 to 1.0 h. After that, the N-C-dots grow rapidly, as their average diameter expands from 2.6 to 15.4 nm within the next 2.0 h. Since then, the growth rate declines and the average size is 16.1 nm as the reaction proceeds for 4.0 h.

Fig. 6 (a–f) TEM images of the N-C-dots prepared at 170 °C in the oxygen flow rate of 50 mL min⁻¹ for 0.5, 1.0, 1.5, 2.0, 3.0, and 4.0 h. Insets in (a–f): the corresponding size distributions of the N-C-dots.

Based on the above observations and previous studies about the formation mechanism of carbogenic materials from small molecules, we propose a possible formation mechanism of the N-C-dots derived from MEA. At first, thermal oxidation of MEA takes place in the presence of oxygen, producing diverse oxidized organic molecules, such as aminoacetaldehyde, glycine, glycollic acid, and oxalic acid, etc.^44,45 Their concentrations increase sharply in the system with heating time. When the concentrations of these oxidized species reach a certain degree, polymerization occurs through intermolecular dehydration,⁴⁶ and simultaneously aromatization of polymers takes place by intramolecular dehydration, leading to the generation of aromatic CN clusters. This stage is so-called the nucleation of the N-C-dots. In the next stage, the nuclei so formed grow outwards by the diffusion of oxidized molecules towards the particle surface. As a result, the particle size increases. Meanwhile, various functional groups (hydroxyl, carbonyl, carboxyl, etc.) passivate the particle surface and facilitate their dispersity in hydrophilic solvents. Thus, the N-C-dots form. However, as the oxidation of alkanolamines is very complicated and the defined structures of the intermediate products are unknown,⁴⁷ great efforts should still be made to clarify the detailed formation process of the N-C-dots in future work.

Effects of oxygen flow rate and reaction temperature on synthesis of N-C-dots

Initially, it was found that heating MEA, a single precursor, in open air brought about a pale yellow dispersion of N-C-dots. However, the reaction rate was rather slow for high-efficiency and large-scale synthesis of N-C-dots, as only 0.07 g of the product was obtained when heating 100 mL of MEA at 170 °C for 2.0 h. If MEA was heated in N₂ atmosphere, no color change was observed, which indicated that the oxygen in air played a crucial role in synthesis of N-C-dots. When pure oxygen was bubbled into MEA, the reaction rate for production of N-C-dots significantly increased. Fig. 7a show the color variations of N-C-dots solutions in different oxygen flow rates. Rationally, a higher oxygen flow rate led to a quicker color variation. Fig. S6a–c† record the temporal evolutions of UV-vis absorption spectra in the oxygen flow rates of 0, 25, and 100 mL min⁻¹. There are adsorption peaks at ∼280 nm in Fig. S6a–c,† just as those observed in Fig. 5b. The production rate of the N-C-dots can be followed by plotting the change in absorbance at ∼280 nm as a function of heating time (Fig. 7b). Fig. 7b clearly reveals that a higher oxygen flow rate results in a faster production rate of N-C-dots. For an efficient and scalable synthesis, in the oxygen flow rate of 100 mL min⁻¹, one could get 3.36 g of fluorescent N-C-dots with quantum yield of 11.9% by heating 100 mL of MEA at 170 °C for 2 hours. The production rate was improved by nearly 50 times compared with the condition that no pure oxygen was added. It should be noted that the N-C-dots could form at relatively low temperature, for example at 110 °C (Fig. S7a†). However, the N-C-dots fabricated at 110 °C exhibit a broader absorption peak (Fig. S7b†), which indicates that lower temperature leads to broader size distribution of the nanodots. In addition, the reaction rate decreases drastically as the temperature falls (Fig. S7c†). Furthermore, the quantum yield of the N-C-dots fabricated at 110 °C never reaches 3%, much lower than that synthesized at 170 °C. Therefore, a higher temperature (170 °C) and a higher oxygen flow rate (100 mL min⁻¹) are more favorable for the synthesis of bright N-C-dots with uniform size on a large scale.

Fig. 7 (a) Photographs of the N-C-dots prepared at different reaction time (from left to right: 0, 10, 30 min, 1.0, 1.5, 2.0, 3.0, and 4.0 h) and in different oxygen flow rates (from down to up: 0, 25, 50, and 100 mL min⁻¹) The temperature was fixed at 170 °C. (b) Time dependence of the absorbance at the peak position (∼280 nm).

Effects of hydroxy and amino groups on synthesis of N-C-dots

In order to investigate the effects of hydroxy and amino groups of alkanolamines on synthesis of N-C-dots, besides MEA, two other representative alkanolamines, DEA and TEA, were selected as precursors. Reasonably, alkanolamines containing more hydroxy and amino groups have higher boiling points (the boiling points of MEA, DEA, and TEA are 170, 271, and 335 °C, respectively), and afford to synthesize N-C-dots at higher temperatures with faster production rates. For example, heating 100 mL of TEA at 270 °C in the oxygen flow rate of 100 mL min⁻¹ for 2 hours, 19.12 g of N-C-dots was obtained. The production yield of N-C-dots was improved by about 6 times compared with heating 100 mL of MEA at 170 °C in the oxygen flow rate of 100 mL min⁻¹ for 2 hours. It is found that the production rate of N-C-dots from MEA, DEA, and TEA in the same synthetic conditions is TEA > MEA > DEA (Fig. S8†). The DEA- and TEA-derived N-C-dots also possess absorption peaks at about 280 nm and show excitation wavelength dependent PL properties (Fig. S9†), which are similar with those of MEA-derived N-C-dots.

It should be mentioned that we can also produce N-C-dots from ethylenediamine (Fig. S10†), which only contains two amino groups. However, because of the lack of hydroxy groups, the boiling point of ethylenediamine (116 °C) is much lower than those of alkanolamines, which limits its fast conversion to N-C-dots. Another organic compound, ethylene glycol (the boiling point of ethylene glycol is 197 °C), containing two hydroxy groups, can convert to nitrogen-free C-dots at relatively high temperature (Fig. S11†). Unfortunately, the production rate of C-dots synthesized from ethylene glycol was rather slow even at 197 °C in the oxygen flow rate of 100 mL min⁻¹. Thus, compared with ethylenediamine and ethylene glycol, alkanolamines which have both high boiling points and easy oxidation property, are more suitable for efficient and scalable production of N-C-dots with oxygen-driven method.

Two-photon cellular imaging

Most previously reported bioimaging applications of C-dots or N-C-dots used one-photon UV or blue light for excitation, which were harmful to biological systems. Two-photon florescence imaging has advantages such as minor autofluorescence background, less photodamage, and larger imaging depth.³ The MEA-derived N-C-dots possess efficient up-conversion PL property, which originates from two or multiphoton active process. This prompts us to explore their potential in two-photon florescence imaging. Fig. 8 show the confocal laser scanning microscopy images of Hela cells which are incubated with the N-C-dots in a concentration of 100 μg mL⁻¹ for 1 h under NIR excitation at 760 nm. Apparently, the N-C-dots have successfully penetrated the membranes in such a short time (1 h) and brightly lightened the whole cells by their two-photon induced florescence. MTT assay was performed to evaluate the cytotoxicity of the N-C-dots (Fig. S12†). The result reveals that the N-C-dots are low-toxic, since more than 80% of the Hela cells remained alive even treated with the N-C-dots in a high concentration of 600 μg mL⁻¹ for 48 h. The fast cellular uptake, strong two-photon florescence, and low cytotoxicity render the N-C-dots attractive probes for two-photon imaging in biological and biomedical applications.

Fig. 8 Two-photo cellular imaging under (a) bright-field and (b) 760 nm excitation. (c) The merged image of (a) and (b).

Conclusions

In conclusion, we have developed an oxygen-driven method for low-cost, large-scale and high-efficiency production of fluorescent N-C-dots using various alkanolamines as both precursors and solvents via a thermal treatment process. The production rate of N-C-dots is remarkably improved by bubbling pure oxygen into alkanolamines. Precise size control of N-C-dots can be achieved by simply changing the reaction time. The as-prepared N-C-dots with abundant hydrophilic groups are well-dispersed in aqueous media and possess strong down- and up-conversion photoluminescence, high photostability, excellent biocompatibility, and low cytotoxicity. These features make them promising candidates for two-photon cellular imaging and beyond.

Acknowledgements

This work was financially supported by the National Basic Research Program (2010CB732401), National Science Fund for Creative Research Groups (21121091), National Natural Science Foundation of China (20875045).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra16989a

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