High-quality water-soluble luminescent carbon dots for multicolor patterning, sensors, and bioimaging

Abstract

An ingenious method for large-scale fabrication of water-soluble photoluminescent carbon dots (CDs) by a one-step microwave pyrolysis of oxalic acid (OA) and urea is developed. The structure and optical properties of the CDs are characterized by transmission electron microscopy, high-resolution transmission electron microscopy, X-ray diffraction patterns, elemental analysis, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, UV-vis absorption, and photoluminescence spectroscopy. The mechanism for the formation of the CDs is also discussed. In contrast to other CD-based nanomaterials, the as-prepared CDs exhibit high fluorescent quantum yield and excellent stability in both organic and inorganic phases. After simple post-treatment, the CDs are applied as fluorescent powder, showing their promising potential for further wide usage. In addition, the CDs can be utilized as a modification-free biosensor reagent capable of detecting Fe³⁺ and Ag⁺ in complex environments. The linear ranges for Fe³⁺ and Ag⁺ were 1.0–130 and 0.50–200 μM with the corresponding detection limits of 4.8 and 2.4 nM, respectively. More significantly, the CDs are superior fluorescent bioimaging agents in plants and cells based on their excellent water-solubility and ultra-low toxicity. Finally, the as-synthesized CDs are successfully applied for detecting Fe³⁺ and Ag⁺ in biosystems.

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Introduction

Nanomaterials have a tremendous impact on the advancement of a wide range of fields including electronics, photonics, energy, catalysis, and medicine. In the development of fluorescent nanomaterials, the discovery of semiconductor quantum dots (QDs) is considered as a major milestone. However, a major disadvantage limits the use of QDs because most of the high-performance QDs are composed of toxic heavy metal elements such as cadmium. In this regard, carbon nanomaterials have fascinating optical properties and already show encouraging performance in bioimaging and biosensing. Fluorescent carbon nanomaterials appear in different forms such as fullerene,¹ carbon nanotubes,² nanodiamonds,³ fluorescent graphene,^4,5 and a rising star: carbon dots (CDs)^6–9 which were accidently discovered as a by-product during the electrophoretic purification of single-wall carbon nanotubes fabricated by arc-discharge methods.¹⁰ Among all these materials, CDs containing heteroatoms have generated especially a lot of excitement and been actively pursued as they are considered the most promising candidate to complement carbon in materials applications because of their intrinsic properties such as electronic properties, surface and local chemical reactivity.^11,12 As such, more synthesis methods were developed for the preparation of various types of CDs doped with heteroatoms, lowering the cost, simplifying operation, improving quantum yield (Φ_s), or realizing larger-scale production. The key issue for designing CDs lies in controlling the number of atoms within one CDs core. For this purpose, raw CDs cores can be obtained by two main approaches “top-down” and “bottom-up”, which have been well summarized in different reviews.^13–16 In brief, the former is based on post-treating of nanocarbon broken from various larger carbon structures, while the bottom-up approach includes thermal decomposition, combustion and dehydration of suitable molecular precursors. Generally, these methods involve intricate processes and severe synthetic conditions, and the Φ_s of the obtained CDs is very low with only a few exceptions.

Recently, microwave pyrolysis of carbohydrates solution has become more and more popular because of the low cost and simple synthesis.^8,17–20 However, to achieve highly luminescent CDs, surface-passivation reagents are usually required.^21–23 Microwave pyrolysis of different carbohydrates either in the absence or presence of any surface passivating agent and dehydration of carbohydrates with subsequent surface passivation have been reported to produce CDs with sufficient Φ_s. Maybe because of the high reaction temperature, not increasing slowly from low to high temperature, the polymer-like CDs could change to carbogenic CDs rapidly, producing more satisfactory CDs.⁸ Moreover, much attention has been paid to their applications in biological labelling,^6,7,24 bioimaging,^4,6,7,24 drug delivery,^25,26 sensors,^24,27 and optoelectronic devices.^15,28,29 In principle, of particular interest and significance are the findings that CDs in the solid states can exhibit strong photoluminescence (PL) emission and the PL from CDs can be quenched efficiently by either electron acceptor or electron donor molecules in solutions.⁸ Therefore, exploring efficient strategies for the large-scale fabricating of CDs with high Φ_s and further expanding their novel applications are still a challenge.

Herein, for the first time, using oxalic acid (OA) as carbon source and urea as surface passivated agent, a simple, low cost, and green preparative strategy toward water-soluble CDs with high Φ_s on a large scale by one-step microwave-assisted method is reported. The morphology and chemical structures are investigated extensively. Compared to the currently available CDs-based nanomaterials, our strategies for fabricating CDs hold great promise in their synthesis and applications. The benefits are: (1) simple synthesis requiring only 8 min microwave irradiation, (2) low-cost and large-scale fabrication, thus providing potential for industrial production, (3) strong fluorescence in dry and aggregate states, hence ensuring its innovative applications in patterning and information storage, (4) relatively high Φ_s (28.7%), (5) excellent water-solubility and stability and good biocompatibility, utilizing as fluorescent bioimaging agents in cells and plants, and (6) efficient PL quenching by Fe³⁺ and Ag⁺ ions, providing particularly useful platform for portable detection of Fe³⁺ and Ag⁺ in complex environments.

Experimental

Materials

Oxalic acid, polyvinyl alcohol (PVA), potassium bromide and urea were obtained from Aldrich (Milwaukee, WI, USA). Dimethyl sulfoxide (DMSO), Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), trypsin, ethylenediaminetetraacetic acid (EDTA), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Solarbio (Beijing, China). Other reagents were from Beijing Chemical Reagents Company (Beijing, China). All reagents of analytical reagent grade or above were used as received without further purification. All aqueous solutions were prepared with ultrapure water (≥18.25 MΩ cm) from a Milli-Q Plus system (Millipore, Bedford, MA, USA).

Synthesis of CDs

The CDs was synthesized by a facile green route of microwave-assisted pyrolysis method. Briefly, 0.50 g OA and 0.50 g urea were dissolved with 10 mL ultrapure water in a 100 mL beaker under ultrasonic stirring for 30 min to form a homogeneous solution. Then the reaction mixture was placed at the center of the rotation plate of a domestic microwave oven (700 W) and heated for 8.0 min. The mixture was cooled down to room temperature and 20 mL of ultrapure water was added. The solution was purified by a dialysis membrane tube with MWCO of 500–1000 Da (Spectrum Laboratories, Rancho Dominguez, CA, USA) in a 2 L ultrapure water with stirring and recharging with fresh ultrapure water every 24 h over a course of 3 days. Finally, a clear and light yellow aqueous solution containing CDs was obtained and lyophilized to yield the dry CDs product.

Characterisation

The elemental analysis was carried out on an Elementar Analysensysteme vario EL cube elemental analyzer (Hanau, Germany). Analyses were performed in triplicate and the average values were obtained. The transmission electron microscopic (TEM) and high-resolution transmission electron microscopic (HRTEM) images were acquired on a JEOL JEM-2100 transmission electron microscopy (Tokyo, Japan) with an accelerating voltage of 300 kV. The Fourier transform infrared spectra (FTIR) were recorded on a Bruker Tensor II FTIR spectrometer (Bremen, Germany). The X-ray diffraction (XRD) patterns were acquired on a Bruker D8 Advance powder X-ray diffractometer (Bremen, Germany) with CuKα radiation operating at 40 kV and 40 mA. The data were collected from 2θ = 10–70° at a scan rate of 0.03° per step and 2 s per point. The X-ray photoelectron spectra (XPS) were done on an AXIS ULTRA DLD X-ray photoelectron spectrometer (Kratos, Tokyo, Japan) with AlKα radiation operating at 1486.6 eV. Spectra were processed by the Case XPS v.2.3.12 software using a peak-fitting routine with symmetrical Gaussian–Lorentzian functions. The UV-vis spectra were performed on a Varian Cary 300 Scan UV-vis absorption spectrophotometer at 250–600 nm. The PL spectra were recorded on an Edinburgh FLS 920 spectrofluorometer (Livingston, UK).

Nanosecond fluorescence lifetime experiments were performed using a FLS 920 time-correlated single-photon counting (TCSPC) system under right-angle sample geometry. An Edinburgh EPL 405 nm picosecond diode laser with a repetition rate of 2 MHz was used to excite the samples. The fluorescence was collected by a photomultiplier tube (Hamamatsu H5783p) connected to a Becker & Hickl SPC-130TCSPC board (Berlin, Germany). The time constant of the instrument response function was 50 ns.

Preparation of CDs/PVA composites film

Firstly, 2.0 mL (2.0 mg mL⁻¹) of CDs aqueous solution was mixed with 5.0 mL PVA solution (10 wt%) by slowing shaking in order to obtain uniform CDs/PVA solution. Then the mixture was coated onto a glass substrate and then dried in an oven under 70 °C for 2 h to obtain a CDs/PVA composite film.

Fluorescent sensors for Fe³⁺ and Ag⁺ detection

The freeze-dried CDs powder was dissolved in a phosphate buffer saline (PBS comprising 137 mM NaCl, 2.7 mM KCl, 8.0 mM Na₂HPO₄, and 2.0 mM KH₂PO₄) solution (pH 7.4) with a concentration of 0.50 mg mL⁻¹. Then, aqueous solutions containing seventeen kinds of metal ions, K⁺, Na⁺, Ag⁺, Ca²⁺, Mg²⁺, Zn²⁺, Fe²⁺, Cu²⁺, Ni²⁺, Co²⁺, Pb²⁺, Hg²⁺, Ba²⁺, Cd²⁺, Al³⁺, Cr³⁺, and Fe³⁺, were prepared with a concentration of 10 mM. To evaluate the selectivity of CDs, 0.20 mL of the above metal ion solution was mixed with 1.8 mL of the CDs solution, and then the PL spectra were measured for recording the fluorescence intensity. To evaluate the detection range of Fe³⁺ and Ag⁺, 0.20 mL of the solutions containing different concentrations of Fe³⁺ and Ag⁺ were mixed with 1.8 mL of the CDs solution, respectively. The control sample was prepared by mixing 0.20 mL of ultrapure water with 1.80 mL of the CDs solution. All the samples were excited (λ_ex) at 352 nm, and the intensities of fluorescence emission (λ_em) at 426 nm for each sample were recorded for comparison.

MTT assay

For the cell cytotoxicity test, human cervical carcinoma SiHa cells were first plated on a Costar® 96-well cell culture cluster and cultured at 37 °C with 5.0% CO₂ in air for 3 h to adhere cells onto the surface. The well without cells and treatment with CDs was taken as the zero sets. The medium was then changed with 200 μL of fresh DMEM supplemented with 10% FBS containing CDs and the cells were allowed to grow for another 24 and 48 h, respectively. At least six parallel samples were performed in each group. Cells not treated with CDs were taken as the controls. After adding 20 μL of 5.0 mg mL⁻¹ MTT reagent into every well, the cells were further incubated for 4 h. The culture medium with MTT was removed and 150 μL of DMSO was added. The resulting mixture was shaken for ca. 10 min at room temperature. The optical density (OD) of the mixture was measured at 490 nm with a SunRise microplate reader (Tecan Austria GmbH, Grödig, Austria). The cell viability was estimated as: cell viability (%) = (OD_treated/OD_control) × 100%, where OD_control and OD_treated were obtained in the absence and presence of CDs, respectively.

Multicolor cellular imaging

The human cervical carcinoma SiHa cells were cultured in DMEM supplemented with 10% FBS and the cells were seeded in the culture dish and cultured with 0.50 mg mL⁻¹ CDs, 0.50 mg mL⁻¹ CDs/1.0 mM Fe³⁺ and 0.50 mg mL⁻¹ CDs/1.0 mM Ag⁺, respectively. After incubation at 37 °C for 12 h, the SiHa cells were harvest using 0.25% trypsin/0.020% EDTA, washed three times (1.0 mL each) with pH 7.4 PBS and kept in PBS for optical imaging by a Olympus FV1000 confocal microscope (Tokyo, Japan) with 20× and 40× objective.

Results and discussion

The synthetic method can be used to prepare different types of CDs by tuning the mole ratio of the precursors to the surface passivated agent as depicted in Fig. S1A.† Various optical determinations were employed to characterize the as-synthesized CDs. As all the CDs products synthesized under different reaction conditions possess similar spectral characteristics, the CDs product synthesized with 8.0 min microwave irradiation and 0.67 mole ratio of COOH/NH₂ possessed the highest Φ_s and was chosen to perform other tests unless otherwise stated. Reaction time is another important parameter for CDs synthesis. Fig. S1B† depicts the Φ_s of CDs synthesized at various reaction times. As the reaction time proceeds from short to long, the polymer-like CDs are converted into carbogenic CDs. In a modest reaction time, polymer-like CDs are formed and the PL arises from the surface/molecule state (perhaps owing to amide-containing fluorophores). In a short or long reaction time, owing to further carbonization, partial carbogenic CDs are formed and the PL is derived from the synergistic effect of the carbogenic core and the surface/molecule state. The carbogenic core plays a greater role in CDs no matter the synthesis time decreases or increases. The optimal reaction time is found to be 8.0 min as it produces the highest Φ_s.

The CDs were prepared by the microwave-assisted method which has described in the experimental section (Fig. 1 and S2†). The reaction was conducted by first condensing OA and urea, whereupon they formed polymer-like CDs, which were then carbonized to form the CDs.^16,24

Fig. 1 A synthetic route using oxalic acid and urea from ionization to condensation, polymerization, and carbonization.

The morphology and structure of CDs were confirmed by further analysis. The TEM image shows that the synthesized CDs are highly monodisperse and narrowly distributed with diameters in the range of 3.25 ± 0.2 nm (Fig. 2A and top inset). In addition, a magnified TEM image of CDs (bottom inset of Fig. 2A) and the representative HRTEM image of CDs (Fig. S3†) show that the lattice spacing is ca. 0.20 nm which is consistent with the (100) facet of graphitic structure. A typical XRD pattern of CDs displays two characteristic diffraction peaks located around 22.2° (0.227 nm) and 27.8° (0.648 nm) (Fig. S4†), suggesting an amorphous nature which is consistent with the discernible lattice structures from the inset in TEM image and HRTEM image.

Fig. 2 (A) TEM image, particle size distribution (top inset) and magnified TEM image (bottom inset) of the as-synthesized CDs. (B) XPS survey scan of CDs and the inset displays the C1s XPS. (C) FTIR spectrum of CDs.

To probe the chemical composition and content of the as-synthesized CDs, the elemental analysis and XPS measurements were obtained. Table S1A† summarizes the elemental analysis of CDs which is consistent with the results of XPS (vide infra). The doping concentration of N is about 29.86%, which is much higher than those reported before with other methods and precursors.^30–32 As shown in Fig. S5,† it is observed that the appropriate nitrogen content in CDs can improve the Φ_s of CDs. The elemental contents are expressed in terms of relative number of atom as depicted in Table S1B.† The empirical formula for CDs is approximately C₆H₁₇N₇O₉. As shown in Fig. 2B, the survey XPS of CDs sample reveals the presence of C, N and O as well as limited H without any other impurities. The binding energy peaks at 286.3, 397.3 and 528.8 eV correspond to C1s, N1s, and O1s, respectively. In detail, the C1s spectrum (inset of Fig. 2B) are deconvoluted into four peaks at 284.98, 286.48, 288.45 and 289.43 eV, which are attributed to C–C/C=C, C–O, N–C=N, and O–C=O, respectively.^33–36 The N1s spectrum (Fig. S6A†) demonstrates three peaks at 399.34, 400.24 and 401.24 eV, which are associated with C–N–C, N–(C)₃ and C=N–C functionalities, respectively. The O1s of CDs spectrum (Fig. S6B†) shows two peaks at 531.75 and 533.05 eV attributing to the C=O and C–OH/C–O–C groups, respectively. In addition, IR spectra were recorded to identify the functional groups on CDs. As shown in Fig. 2C, the strong peaks at 1719, 1609, 1429, 1321, and 1149 cm⁻¹ corresponding to C=O, C=C stretching, CH₂, amido C–N and asymmetric stretching vibrations of C–NH–C, respectively. The broad peaks centered at 3408–3454, 3209–3348, 3013–3047, and 2794–2858 cm⁻¹ are attributed to the O–H vibration stretch of the carboxylic moiety, N–H stretch of the amine groups, stretching vibration of C–OH, and C–H, respectively, which improve the hydrophilicity and stability of the CDs in aqueous system.³³ For OA (Fig. S7A†), two characteristic peaks ascribed to –OH and C=O stretch are observed. For urea (Fig. S7B†), two characteristic peaks attributing to N–H bending (1466 cm⁻¹) and C–N stretching (1153 cm⁻¹) of amines are identified. The IR spectra suggest that the possible amidation reaction should have taken place between OA and urea and the CDs surface is covered with the amine, amide and carboxylic acid groups.

In quest of exploring the optical properties of the as-prepared CDs, the UV-vis absorption and PL spectra are acquired and depicted in Fig. 3. The absorption spectrum shows a prominent peak at 350 nm assigned to n → π* transitions of C=O bonding. The optimal excitation and emission wavelengths are located at 352 and 426 nm respectively, indicating the narrow size distribution of CDs as well. The photograph of the CDs dispersion under UV light (365 nm) exhibits a blue color (inset of Fig. 3A), further revealing that the resultant CDs exhibit blue fluorescence. To further explore the optical properties of the as-prepared CDs, the excitation-dependent PL behavior was conducted and depicted in Fig. 3B. The PL spectra are bathochromically shifted with the increase in the λ_ex, indicating that the PL band can be tuned by adjusting the λ_ex. The λ_em are red-shifted from 405 to 495 nm for the CDs when the λ_ex moves from 280 to 460 nm. The λ_ex-dependent PL behavior is common with CDs.^{17,19,24,37,38} This behavior is contributed to the surface state affecting the band gap of CDs. The surface state is analogous to a molecular state whereas the size effect is a result of quantum dimensions, both of which contribute to the complexity of the excited states of CDs.³⁹ Fortunately, λ_ex-dependent PL behavior can be useful in multicolor imaging applications (vide infra). Furthermore, the Φ_s is 28.7% for CDs (Fig. S8A†) using quinine sulfate (Fig. S8B†) as the reference. Besides high Φ_s, the as-prepared CDs exhibit excellent stability which is essential for practical applications. The PL stability of CDs to the effects of the ionic strength (in terms of the concentration of KCl) and pH of solutions was investigated. There were no changes in PL intensity or peak characteristics under different concentrations of KCl (Fig. S9A†), which is significant because it is necessary for CDs to be used in the presence of physical salt concentrations in practical applications. Another interesting phenomenon is the pH-dependent PL behavior (Fig. S9B†). The PL intensities decrease in a solution of high or low pH, but remain constant in a solution of pH 5.0–10. Since the PL intensity is highest and maintains fairly constant at the physiological pH 7.0–7.5, it has potential for application in cellular imaging (vide infra). Fig. S9C† depicts the photostability of the CDs at λ_ex/λ_em of 352/426 nm. No significant change in PL intensity was observed (>91%) after 200 min exposure to a 75 W xenon arc light. The dry CDs powder sample could be repeatedly re-dispersed in water without any aggregation, which is advantageous for preservation and transportation. The obtained CDs solution exhibits homogeneous phase without any noticeable precipitation at ambient conditions for six months, indicating their long-term colloidal stability. The preeminent PL of CDs makes the feature of their great potential applications, which will be discussed in the next section.

Fig. 3 (A) UV-vis absorption, PL excitation (λ_ex) and emission spectra. The inset displays photographs of the CDs under daylight (left) and UV irradiation (right) in aqueous solution. (B) PL spectra of CDs at different λ_ex 280–460 nm. The concentration of CDs is 0.50 mg mL⁻¹.

Highly-dense immobilization of CDs into flexible organic thin films is an indispensable procedure for their wide applications. Thus, we extend the use of CDs as for fabrication of fluorescent film. The CDs was blended into a compatible PVA solution to form a thin film by coating on a glass plate and dried at 70 °C. Fig. 4A and B show photographs of the glass with the CDs-doped fluorescent film under room light (Fig. 4A) and under 365 nm irradiation (Fig. 4B). It could be seen that the CDs-doped fluorescent film owns certain transparency under visible light and blue fluorescent under 365 nm irradiation, allowing that the CDs/PVA composite film could be used as a security feature.

Fig. 4 The CDs/PVA fluorescent film in the room light (A) and under 365 nm UV irradiation (B). Photographs of CDs powder in the daylight (C) and under 365 nm UV irradiation (D). A CDs-formed fluorescent fingerprint on filter paper captured under a UV lamp (E).

In addition, to make full use of the high Φ_s, the CDs were utilized to prepare CDs solid powder by lyophilizing CDs solution. As seen in Fig. 4C and D, CDs powder owns light yellow under daylight (Fig. 4C) and blue fluorescence under 365 nm irradiation (Fig. 4D). Notably, the fluorescent properties of the CDs powder have been preserved for more than 6 months without significant changes. Furthermore, the CDs could be used as inkpad. As shown in Fig. 4E, a fluorescent CDs fingerprint was printed on a filter paper, which could reflect human's fingerprint discriminately and clearly. Compared with the traditional inks, our water-soluble CDs ink are clear, permanent, adelomorphic, pollution-free and easy to clean with water, providing a new way to feed the potential requirement on a commercial scale.

Of particular interest and significance is the finding that the as-prepared CDs can be utilized as a highly efficient nanoprobe for Fe³⁺ and Ag⁺ detection. Fe³⁺ and Ag⁺ ions can be detected with CDs via luminescence measurements, but those published results suffered from the narrow ranges of detection concentrations,^40–42 low accuracy^43–45 or low selectivity.^24,46 Fig. 5A and B show the fluorescent quenching of CDs at various concentrations of Fe³⁺ (Fig. 5A) and Ag⁺ (Fig. 5B). The insets show the linear relationship between PL intensity and Fe³⁺ and Ag⁺ concentration, respectively. The ratios of F₀/F have good linear correlation with the Fe³⁺ and Ag⁺ concentrations in the range of 1.0–130 μM and 0.50–200 μM, respectively which are much larger than those of the recent reports.^24,40–42 Here F₀ and F are the fluorescent intensities of CDs at 426 nm in the absence and presence of Fe³⁺ or Ag⁺ ions, respectively. The quenching efficiency is fitted by the Stern–Volmer equation, F₀/F = 1 + K_sv[Q], where K_sv is the Stern–Volmer quenching constant and [Q] is the Fe³⁺ or Ag⁺ concentration.⁴⁷ The K_sv are calculated to be 7.0 × 10³ L mol⁻¹ and 8.0 × 10³ L mol⁻¹ with a correlation coefficient r² of 0.996 and 0.992, respectively. The detection limit is estimated to be 4.8 nM and 2.4 nM for Fe³⁺ and Ag⁺ at a signal-to-noise ratio of 3, respectively.⁴⁸ Besides sensitivity, selectivity is another important parameter to evaluate the performance of the sensing system. The effect of representative metal ions (K⁺, Na⁺, Ag⁺, Ca²⁺, Mg²⁺, Zn²⁺, Fe²⁺, Cu²⁺, Ni²⁺, Co²⁺, Pb²⁺, Hg²⁺, Ba²⁺, Cd²⁺, Al³⁺, Cr³⁺ and Fe³⁺) on CDs fluorescence quenching under the same conditions are investigated. As shown in Fig. 5C, most of those metal ions of ultrahigh concentrations (1.0 mM) do not induce significant decrease in PL intensity of CDs except for Fe³⁺ or Ag⁺ ions. The possible mechanism was shown in Fig. S10.† The possible reasons for the high selectivity are attributing to the coordination between the Fe³⁺ or Ag⁺ ions and the phenolic hydroxyl and/or amine functionalities on the CDs, resulting in a nonradiative electron transfer from the excited state of the CDs to the metal ions. The above results clearly prove that our CDs are very promising for Fe³⁺ and Ag⁺ detection in practical applications.

Fig. 5 (A) Fluorescence quenching of CDs in the presence of Fe³⁺ ions (0.0–300 μM). The inset displays the Stern–Volmer plot of CDs at various concentrations of Fe³⁺ (1.0–130 μM) where F₀/F are the PL intensities of CDs (0.50 mg mL⁻¹) in the absence and presence of Fe³⁺, respectively. (B) Fluorescence quenching of CDs in the presence of Ag⁺ ions (0.0–300 μM). The inset displays the Stern–Volmer plot of CDs at various concentrations of Ag⁺ (0.50–200 μM) where F₀/F are the PL intensities of CDs (0.50 mg mL⁻¹) in the absence and presence of Ag⁺, respectively. (C) Comparison of fluorescence intensities of CDs (0.50 mg mL⁻¹) after the addition of Fe³⁺ (1.0 mM), Ag⁺ (1.0 mM) and other different metal ions (1.0 mM). (D) Fluorescence decay of CDs (0.50 mg mL⁻¹) without and with Fe³⁺ (10 mM) and Ag⁺ (3.0 mM) as a function of time at excitation/emission wavelengths (λ_ex/λ_em) of 405/426 nm. IRF is the instrumental response function curve.

Fig. S11† shows the CDs before and after quenching by Fe³⁺ or Ag⁺. It is very obvious that the blue emission weakens after adding Fe³⁺ or Ag⁺ to the CD solution. Fe³⁺ ion is indispensable for a large number of living systems and plays an important role in many biochemical processes, while Ag⁺ ion can cause serious and permanent damage to human organs due to its accumulative characters in the environment and biota. The detection of Fe³⁺ and Ag⁺ ions through a visible fluorescent method would be of considerable benefit. Finally, the fluorescence decay dynamics was measured to investigate the PL quenching mechanism of the system. The fluorescence decay curves of CDs, CDs/Fe³⁺ and CDs/Ag⁺ can be fitted by a double-exponential formula,⁴⁹ involving the lifetimes τ₁ and τ₂. As shown in Fig. 5D and Table S2,† the average lifetime of CDs is 5.41 ns. After coordination with Fe³⁺ and Ag⁺ ions, the lifetime decreases to 4.14 and 2.14 ns, respectively. The decrease in the lifetime indicates an ultrafast CDs/Fe³⁺ and CDs/Ag⁺ electron-transfer process and leads to dynamic quenching. These results indicate the special coordination interaction between Fe³⁺ or Ag⁺ ions and the phenolic hydroxyl groups of the CDs contributing to the quenching progress, which has been used for the detection of metal ions or colored reactions in traditional organic chemistry.⁵⁰

Optical and fluorescent images of bean sprouts grown with CDs aqueous solution (5.0 mg mL⁻¹) under (B) daylight and (C) 365 nm irradiation. Laser scanning confocal microscopy images of SiHa cells incubated with (D) 0.50 mg mL⁻¹ CDs, (E(i)) 0.50 mg mL⁻¹ CDs and 1.0 mM Fe³⁺, and (E(ii)) 0.50 mg mL⁻¹ CDs and 1.0 mM Ag⁺ at 37 °C for 12 h. The first left panels show the bright-field images of SiHa cells. The second, third and fourth panels are cell images taken at λ_ex/λ_em of 405/422 ± 25, 488/500 ± 25 and 543/650 ± 25 nm, respectively. The fifth panels are the merged images of the second and third panels. The sixth panels are the merged images of the second and fourth panels. The seventh panels are the merged images of the third and fourth panels.

These encouraging results prompted us to evaluate the feasibility of CDs as a new fluorescent marker for living cell imaging.^7,8,51 For effective bioimaging, it is required that the selected fluorescent marker possesses not only optical merits but also low cytotoxicity.^7,8,51,52 To evaluate the cytotoxicity of CDs, the viability of human cervical carcinoma SiHa cells treated with CDs measured by the MTT method. As shown in Fig. 6A, SiHa cells were incubated with different doses of CDs for 24 and 48 h, respectively. The viability of the cells remained greater than 85% even incubated with ultrahigh concentration (800 μg mL⁻¹) of CDs for 48 h, demonstrating instinctively low toxicity of the CDs (without any further functionalization).⁹

Fig. 6 (A) Cytotoxicity test of CDs on SiHa cells viability. The values represent percentage cell viability (mean% ± SD, n = 6).

Given the low cytotoxicity, we further tested the toxicity and feasibility of CDs as bioimaging agents in both plants and in vitro imaging. Firstly, some selected mung beans were raised in aqueous solution of CDs (5.0 mg mL⁻¹) at 25 °C. Similar to bean sprouts grown in water (Fig. S12†), no abnormal sprouts were found after three days grown in aqueous solution of CDs (Fig. 6B). Under UV irradiation, sprouts exhibited strong characteristic blue luminescence, illustrating that CDs could permeate throughout the plant cells but were nontoxic and did not hinder plant growth (Fig. 6C). The potential of CDs as luminescent probes for in vitro imaging was then carried out on SiHa cells. Fig. 6D displays photographs of the SiHa cells captured by a laser scanning confocal microscope (LSCM). It is obvious that the transfected SiHa cells became quite bright owing to the strong fluorescence from CDs, indicating a large amount of CDs have been internalized into the cells. Consistent with fluorescence microscopy assay results, bright blue, green and red fluorescence could be observed under ultraviolet, blue and green light excitation, and the every two-channel luminescence images for the same scanning area overlapped well. This multicolor emission shows a great advantage of the CDs over other labeling agents because it gives us much space to choose the wavelength for observation.

In addition, the bright-field images of the SiHa cells incubated with (i) CDs/Fe³⁺ and (ii) CDs/Ag⁺ (first panels in Fig. 6E) indicate clearly the normal morphology of the cells, verifying that CDs/Fe³⁺ and CDs/Ag⁺ are biocompatible and possess minimum toxicity to the cells. The cells display blue (second panels in Fig. 6E), green (third panels in Fig. 6E), and red (fourth panels in Fig. 6E) emissions when they are excited with 405, 488, and 543 nm lasers, respectively. The cells incubated with CDs/Fe³⁺ and CDs/Ag⁺ emit weaker than that of CDs, attributing to the fact that Fe³⁺ and Ag⁺ could quench CDs in aqueous solution (vide supra). As such, CDs can be an effective probe for monitoring Fe³⁺ and Ag⁺ in biosystem.

Conclusions

For the first time, CDs have been synthesized from a fast, simple and “green” route with OA as carbon source and urea as surface passivated agent with a Φ_s as high as 28.7%. The morphology and chemical structures have been investigated extensively by (HR)TEM, XRD, XPS, FTIR, UV-vis absorption, and PL spectroscopy. Moreover, CDs show good sensitivity and selectivity to Fe³⁺ and Ag⁺ in complex environments, and has a remarkable selectivity over other representative metal ions, it can serve as an effective probe for PL detection of Fe³⁺ and Ag⁺ with detection limits as low as 4.8 and 2.4 nM, respectively. Furthermore, a particularly attractive feature of the prepared CDs is their excellent solubility and ultra-low toxicity, empowering the CDs for plant use and in vitro imaging. Notably CDs could be utilized as a reagent capable of detecting Fe³⁺ and Ag⁺ in biosystem. Combining its simple and fast synthetic method, favorable optical properties, low cytotoxicity and ease of labelling, it is anticipated that CDs could have potential applications in biological labelling, disease diagnosis and biosensors.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (21175086, 21175087).

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Footnotes

† Electronic supplementary information (ESI) available: Effect of precursors: n_COOH/n_NH2 and reaction time on quantum yield of CDs, picture of CDs sample, HRTEM image of CDs, XRD pattern of CDs, elemental analysis, nitrogen's content in CDs on quantum yield of CDs, N1s and O1s XPS of CDs, IR spectra of OA and urea, plots of integrated PL intensity against absorbance of CDs and quinine sulfate, effect of ionic strength and pH on PL intensity of CDs, the time-dependence of fluorescent intensity of CDs, sensing principle of the CDs based probe for Fe³⁺ and Ag⁺, photographic images of CDs before and after adding Fe³⁺ and Ag⁺ under daylight and UV irradiation, and photographs of a bean sprout grown with water under daylight. See DOI: 10.1039/c4ra16233a

‡ These authors contributed equally to this work.

§ Present address: Acadia Divinity College, Acadia University, 15 University Avenue, Wolfville, Nova Scotia, B4P 2R6, Canada.

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