Microwave-assisted facile synthesis of yellow fluorescent carbon dots from o-phenylenediamine for cell imaging and sensitive detection of Fe³⁺ and H₂O₂

Abstract

Strongly yellow fluorescent carbon dots (CDs) have been directly synthesized from o-phenylenediamine (o-PD) through a facile microwave-assisted method. The as-prepared o-CDs exhibit excitation-dependent photoluminescent behavior and excellent water-solubility due to some amino or hydroxy functional groups on the surface. An emission peak appears at 573 nm when the o-CDs solution is excited at 400 nm, and the quantum yield (QY) is 38.5%. Owing to their low toxicity and water solubility the as-prepared o-CDs can be directly used for cell imaging. More importantly, the as-prepared o-CDs solution also shows sensitivity for H₂O₂. The limits of detection (LOD) for Fe³⁺ and H₂O₂ are 16.1 nM and 28.1 nM, respectively, which is much lower than what's reported in previous studies. The fluorescence intensity also shows a dependence on pH and the strongest fluorescence intensity appears at pH 9. In addition, we also find that the fluorescent properties of CDs prepared from m-phenylenediamine (m-PDs) and p-phenylenediamine (p-PDs) are quite different from those of o-CDs.

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1. Introduction

In recent years, fluorescent nanoparticles have been paid more and more attention due to their prominent optical properties and potential applications in biology, medicine etc.^1–3 At present, semiconductor quantum dots and carbon nanoparticles are two kinds of fluorescent materials that have been extensively studied.^4–6 However, semiconductor quantum dots are highly toxic because of their heavy metals, such as Cd, Pb, Cu etc.^7–9 In contrast, carbon dots (CDs), as one kind of carbon nanoparticle, are attracting great interest owing to their low toxicity, low cost, biocompatibility, and excellent photoluminescence.^10–12 CDs are usually composed of nano-sized sp² hybridized graphitic cores and carbonyl surface moieties,¹³ and they can be widely used in the field of biochemical sensing,^14,15 florescent probes,^16,17 photocatalytic technology,^18,19 environment testing,²⁰ drug carriers,^21,22 etc.

In the field of biological imaging of CDs, short wavelength light has a weak tissue penetration ability owing to the strong tissue absorption of short wavelength light below 600 nm, which limits its application in vivo.^23,24 Additionally, the near-infrared fluorescence (NIR) spectral range (700–1100 nm) and the red fluorescence region (600–700 nm) are generally referred to as the “optical window” of the biological tissues, which lacks efficient endogenous absorbers and gives rise to the minimum of the light scattering, absorbance, and autofluorescence of tissues.²⁵ Therefore, it is highly desirable to prepare CDs whose excitation and emission peaks are able to be closer to or brought into the “optical window” for in vivo optical bioimaging of deep tissues.

Phenylenediamine, which has three kinds of isomers: o-phenylenediamine (oPD), m-phenylenediamine (mPD), and p-phenylenediamine (pPD), has been used as the precursor for the synthesis of CDs.²⁶ Recently, Jiang et al. synthesized red, green and blue fluorescent carbon dots (CDs) using three different phenylenediamine isomers in ethanol through a solvothermal method.²⁷ In their approach a high temperature (∼180 °C) and a long reaction time (∼12 h) are needed. In comparison with the time consuming hydrothermal methods, microwave-assisted approach is regarded to be energy efficient for the synthesis of CDs, and the reaction time is usually 10–20 minutes.^28–30

Herein, we have successfully prepared o-CDs with strongly yellow fluorescence through a microwave-assisted facile method using o-phenylenediamine (oPD) as the carbon precursor. Different from the reported solvothermal method, the microwave-assisted approach shows a high efficiency and the reaction process only takes 20 minutes with water as the reaction medium. In addition, we also compare the fluorescent property of the CDs prepared from m-phenylenediamine (m-PDs) and p-phenylenediamine (p-PDs), respectively, and find that they are quite different from that of o-CDs. The aqueous solutions of o-CDs and m-CDs emit strongly yellow and viridescent fluorescence under 365 nm-UV light, respectively. But no obvious fluorescence is observed for the aqueous solution of p-CDs. Owing to high QY, low toxicity and water solubility the as-prepared o-CDs can be directly used for cell imaging. More importantly, the optical properties and formation mechanism of the o-CDs have been investigated, and they have also been applied to detect metal ions, whose contents in biological system are very crucial for some human diseases,³¹ pH value, which is an important parameter in diverse physiological processes,^32–34 and H₂O₂, which can act as a canonical mark for oxidative damage to living body.³⁵ The as-prepared o-CDs solution is also sensitive to H₂O₂. The limit of detection for Fe³⁺ and H₂O₂ is 16.1 nM and 28.1 nM, respectively, which are much lower than what's reported in previous studies. Additionally, the fluorescence intensity of the as-prepared o-CDs also shows a dependence on pH and the strongest fluorescence intensity appears at pH = 9.

2. Experimental section

2.1 Chemicals and materials

All chemicals were purchased from Sigma-Aldrich, unless otherwise mentioned. All glassware was washed with aqua regia (conc. HCl : conc. HNO₃, volume ratio = 3 : 1), and then rinsed with ultrapure water and ethanol. Ultrapure water was made from ultrapure water purifier (Mini-Q). Cell Counting Kit (CCK-8 kit) was purchased from BOSTER. Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS) and trypsin–ethylene diamine tetraacetic acid (trypsin–EDTA) were obtained from Gibco. SW480 cells were purchased from Cell Resource Center, IBMS, CAMS/PUMS.

2.2 Characterization

All fluorescence measurements were performed on a Flouromax-4 spectrofluorometer (HORIBA Jobin Yvon Inc) with excitation slit set at 25 nm band passes in 1 cm × 1 cm quartz cell. In addition, UV-vis absorption was characterized on a UV5800 spectrophotometer. Transmission electron microscopy (TEM) was examined on JEM-1200EX electron microscopy with an accelerating voltage of 100 kV. Atomic force microscopy observation was performed on Agilent 5500 (Agilent, USA). The FTIR spectra and Raman spectrum were recorded on an IFS 66V/S (Bruker) IR spectrometer in the range of 600–4000 cm⁻¹ and a Nicolet iS10 FT-IR spectrometer. X-ray photoelectron spectroscopy (XPS) analysis was measured on an ESCALAB MK II X-ray photoelectron spectrometer using Mg as the exciting source. The mass spectrum data was obtained by a MALDI TOF/TOF mass spectrometer. Quantum yield (QY) was measured using rhodamine 6G in 0.05 M sulfuric acid solution (literature quantum yield 0.54 at 310 nm) as a standard for o-CDs. The optical densities measured on UV-vis spectra were acquired on a UV-3010 spectrophotometer (Hitachi, Japan) using a 1 cm path length cuvette. Absolute values are calculated using the standard reference sample that has a fixed and known fluorescence QY value. The decay curve was recorded on a PR305 phosphorophotometer.

2.3 Synthesis of CDs

0.4 g phenylenediamine was first dissolved in 20 ml ultrapure water, the mixed solution was heated by the microwave with the power of 750 W (MCR-3 microwave chemical reactor) for 20 minutes, and then the products were cooled to room temperature and dissolved by 20 ml ultrapure water. In this way, the original CDs aqueous solution was obtained. After being placed for 1 day, the solution was purified by a 0.22 μm filter membrane. The purified CDs solution were first dried at 60 °C in a constant temperature and humidity drying box, and then 80 °C in a vacuum drying oven.

2.4 Quantum yield

The QY of the as-prepared C-dots can be calculated through the equation below:

In this equation, Q is the quantum yield of our sample and I is the measured intensity of luminescent spectra. A is the optical density at excitation wavelength. QY was measured choosing rhodamine 6G in ethanol (quantum yield is 0.95 at 488 nm) as a standard for o-CDs. The subscript “R” refers to standard sample with known quantum yield and “o-CD” stands for the sample in this equation. To minimize re-absorption effects, the absorbance of o-CDs solution which is in the 10 mm fluorescence cuvette was kept ≤0.05 at the excitation wavelength of 400 nm.

2.5 Detection of metal ions

9 kinds of metal ion aqueous solutions (Ni²⁺, Mg²⁺, Fe²⁺, Ca²⁺, Fe³⁺, K⁺, Cu²⁺, Zn²⁺, Mn²⁺) with a concentration of 5 mM were first prepared. Then, the diluted o-CDs aqueous solution (0.8 mg ml⁻¹) was mixed with the 9 kinds of pre-made metal ions solutions with the ratio of 1 : 9, respectively. The mixed solutions were placed for about 30 minutes at a room temperature and then their PL intensities were examined immediately by a fluorescence spectrophotometer.

2.6 Sensitive detection of Fe³⁺

Fe³⁺ aqueous solutions with a certain concentration gradient from 5 mM to 0 mM were firstly prepared and then mixed with the diluted o-CDs aqueous solution (0.8 mg ml⁻¹) with the ratio of 1 : 9, respectively. The mixed solutions were placed for about 30 minutes at a room temperature and then their PL intensities were examined immediately by a fluorescence spectrophotometer.

2.7 Detection of the sensing of the obtained o-CDs for pH values and H₂O₂

Phosphate buffers with different pH values (pH = 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) were mixed with the diluted o-CDs aqueous solution (0.8 mg ml⁻¹), respectively. The mixed solutions were placed for about 30 minutes at a room temperature and their PL intensities were examined immediately by a fluorescence spectrophotometer.

H₂O₂ aqueous solutions with different concentrations (0–50 μM) were mixed with the diluted o-CDs aqueous solution (0.8 mg ml⁻¹), respectively. The mixed solutions were placed for about 30 minutes at a room temperature and their PL intensities were examined immediately by a fluorescence spectrophotometer.

2.8 Cell culture, cytotoxicity assay and cell imaging

SW480 cells were maintained in DMEM/high glucose (1×) medium supplemented with FBS of 10% (v/v) at 37 °C in 5% CO₂ humidified atmosphere. Exponentially growing cells were dissociated with 0.25% trypsin–EDTA (1×) cell dissociation medium (GIBCO).

The cytotoxicity of o-CDs to SW480 cells was evaluated by the CCK8 assay. Briefly the cells were seeded into a 96-well plate at 100 μL per well for 24 h at 37 °C. After the culture medium was removed, the medium containing o-CDs with different concentrations (2–0.25 mg ml⁻¹) was added into subsequently. The cells were then incubated for another 24 h. After that, CCK8 solution, in the volume of 10 μL per well, was added into each well and incubated at 37 °C for 1 h. Then we used the enzyme standard instrument to measure the absorbance at 450 nm.

The bioimaging was also conducted. The cells digested by the 0.25% trypsin–EDTA treatment were seeded in glass bottomed culture dishes (Mat Tek) and cultured for 24 h, then the cell culture medium in the dishes was discarded, the remainder cells were washed for 3 times with PBS (pH = 7.3), after that, 1 ml o-CDs aqueous solution and 1 ml cell culture medium without FBS were added to different dishes, control group and experimental group respectively, then they were incubated for another 4–6 h in the cell culture incubator. When the dishes cultured cells were carried out, the culture medium in them were removed as well, the remainder cells were washed for 3 times, and then 1 ml paraformaldehyde (4%, Sigma) was added. The dishes were placed in 4 °C for 30 min to fix the state of cells. At last, the fixed cells in dishes can be observed by an NIKON TE2000 inverted fluorescence microscope system.

3. Results and discussion

During the synthesis process of CDs, three isomers of phenylenediamine [o-phenylenediamine (o-PD), m-phenylenediamine (m-PDs) and p-phenylenediamine (p-PDs)] were dissolved in ultrapure water, respectively. Before microwave heating, the aqueous solutions of o-PD and m-PD are all clear and transparent but the aqueous solutions of p-CD is khaki. After microwave heating, the reaction products of o-PD and m-PD become deep-brown dry solids and deep-brown viscous liquid sequentially, and the reaction product of p-PD become brownish red flakes. Afterwards the resulting three kinds of CDs (named o-CDs, m-CDs and p-CDs from o-PD, m-PD and p-PD respectively) were dispersed in ultrapure water to form clear aqueous solutions. The colors of o-CDs, m-CDs and p-CDs aqueous solutions are yellow, light pink and dark red, respectively, as shown in Fig. 1a (left). Fig. 1a (right) shows the fluorescence of the aqueous solutions of o-CDs, m-CDs and p-CDs when they were irradiated under 365 nm-UV light, and from the picture, it can be seen that the aqueous solutions of o-CDs and m-CDs emit yellow and viridescent fluorescence, respectively. But no fluorescence is observed for the aqueous solution of p-CDs. The results indicate that the molecular structure of phenylenediamine isomers has a significant effect on the fluorescence property of the obtained CDs. After micro-wave heating different phenylenediamine isomers might form CDs with different morphology, size and functional groups, which result in the different fluorescence property of CDs. The study of the detailed reason and the emission mechanism is still in progress. The optical property of the water solutions of the as-prepared three kinds of CDs were also studied. Firstly, UV-visible absorption of the o-CDs aqueous solution seen from Fig. 1b clearly shows that the as-prepared o-CDs have absorption band at 300 nm, which is attributed to the n–π* electron transition.³⁰ Fig. 1c shows the fluorescence spectra of o-CDs at different excitation wavelengths, and the strongest emission peak is at 573 nm with the excitation wavelength of 400 nm, such emission peak is independent of excitation wavelengths in the range from 325 nm to 500 nm. The QY of the diluted aqueous solution of o-CDs is 38.5%. Additionally, in Table 1, we compared the properties of different nanomaterials synthesized using isomers of phenylenediamine as one of precursors in previous publications.

Fig. 1 (a) Photographs of o-CDs, m-CDs and p-CDs dispersed in ultrapure water in daylight (left) and under 365 nm UV irradiation (right), (b) UV-visible absorption spectrum of o-CDs, (c) emission spectra of the as prepared o-CDs at different excitation wavelengths.

Table 1 Comparison of the properties of different nano materials synthesized using phenylenediamine as precursors

Nano material	Reactant	Solvent	Synthesis method	Photoluminescent property		QY (%)	Ref.
				λex (nm)	λem (nm)
a o-PD: o-phenylenediamine.b PoPD/RGO: poly(o-phenylenediamine)/reduced graphene oxide.c GO: graphene oxide.d PpPD: poly(phenylenediamine).e pPD: p-phenylenediamine.f PoQDs: poly(o-phenylenediamine) quantum dots.g o-CD: carbon dots synthesized using o-phenylenediamine as the carbon source.h m-CD: carbon dots synthesized using m-phenylenediamine as the carbon source.i p-CD: carbon dots synthesized using p-phenylenediamine as the carbon source.
C dots	o-PD^a	Water	Hydrothermal	420	567	2	38
PoPD/RGO^b composite nanosheets	GO^c and oPD	H2O	Microwave heating				39
PpPD^d nanospheres	pPD^e, NH4S2O8 and HCl	Water	A combination reaction	296	301	27.0	40
PoQDs^f	oPD, (NH4)2S2O8	Water	Hydrothermal treatment	PoQDs-1	PoQDs-1	40.0	41
				380	430
				PoQDs-2	PoQDs-2
				430	500
				PoQDs-3	PoQDs-3
				460	545
				PoQDs-4	PoQDs-4
				500	590
CDs (o-CDs^g, m-CDs^h, p-CDs^i)	oPD, mPD and pPD	Ethanol	Solvothermal	o-CDs	o-CDs	o-CDs 17.6%	27
				457	544
				m-CDs	m-CDs	m-CDs 26.1%
				319	435
				p-CDs	p-CDs	p-CDs
				457	610	—
CDs (o-CDs, m-CDs, p-CDs)	oPD, mPD and pPD	Water	Microwave	o-CDs	o-CDs	o-CDs	This work
				400	573	38.5%
				m-CDs	m-CDs	m-CDs
				361	520	16.72%
				p-CDs	p-CDs	p-CDs
				326	402	—

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Fig. 2 shows TEM images of o-CDs and the inset shows the particle size distribution of o-CDs. The particle size is mainly around about 8–11 nm. Fig. 2b present AFM images of o-CDs. As shown in Fig. 2b, the average thickness of o-CDs is about 0.42 nm indicating this kind of nano materials are almost monolayer.^36,37

Fig. 2 (a) TEM images of o-CDs, the inset shows the particle size distribution of o-CDs, (b) the AFM images of o-CDs.

Fig. 3a and b show the fluorescence spectra of m-CDs at different excitation wavelengths, and the strongest emission peak is at 520 nm with the excitation wavelength of 361 nm. The QY of the diluted aqueous solution of m-CDs is 16.72%. Fig. 3c and d show the fluorescence spectra of p-CDs at different excitation wavelengths, and the strongest emission peak is at 402 nm with the excitation wavelength of 326 nm, but the fluorescence intensity of the diluted aqueous solution of p-CDs is too low to detect its QY. The QY of o-CDs is much higher than m-CDs and p-CDs, and o-CDs as a consequence were chosen for the following studies. The emission peak of nanoparticles is correlated with their size, shape and functional groups.^27,42

Fig. 3 (a) UV-visible absorption spectrum of m-CDs, (b) emission spectra of the as prepared m-CDs at different excitation wavelengths, (c) UV-visible absorption spectrum of p-CDs, (d) emission spectra of the as prepared p-CDs at different excitation wavelengths.

To identify functional groups existing in the as-prepared o-CDs, FTIR spectroscopy was studied.^43,44 As shown in Fig. 4a, a double peak at 3387 cm⁻¹ and 3366 cm⁻¹ reveals the existence of amino functional groups on the surfaces of the o-CDs and another double broad peak at 3291 cm⁻¹ and 3198 cm⁻¹ demonstrates the existence of –OH groups.^16,38 Moreover, some peaks at 1632–1592 cm⁻¹, 1502–1459 cm⁻¹, 1336 cm⁻¹, 1275 cm⁻¹, 1157 cm⁻¹ and 753 cm⁻¹ reveal that the o-CDs contain such groups as C=O, C=C, C–N= and –CH, and the peaks at about 1056–1032 cm⁻¹ and 928 cm⁻¹ suggest the existence of C–O/C–O–C groups,²⁷ which indicates that there are a lot of unsaturated carbon bonds formed during the carbonization process. Additionally, Raman spectroscopy was also utilized to characterize the intrinsic structure of the o-CDs by Fig. 4b. In the 1000–2000 cm⁻¹ region, the G band peaking at 1848 cm⁻¹ indicates sp² carbon networks and the D-band peaking at 1402 cm⁻¹ reflects the disorder or defects in the surface structure of carbon dots. The existence of a large fraction of defects mainly results from surface oxidation, which is consistent with the FTIR results.⁴⁴

Fig. 4 (a) The FTIR spectrum of the as-prepared o-CDs, (b) the Raman spectrum of the as-prepared o-CDs.

The XPS spectra measurement was also performed to confirm the composition of o-CDs.⁴⁵ Fig. 5a revealed three peaks at 284.82 eV, 398.89 eV, and 532.3 eV, which are attributed to C_1s, N_1s and O_1s, respectively,^7,15 and the ratio of C, N and O in the o-CDs to be 75.14%, 14.93% and 9.93%, respectively. The C_1s spectra (Fig. 5b) showed three peaks at 284.6 eV, 286.0 eV and 287.5 eV, respectively, which are attributed to C–C/–CH, C–N and C=O bands in o-CDs. The N_1s spectra (Fig. 5c) showed three peaks at 398.7 eV, 399.5 eV and 400.3 eV, respectively, which are attributed to the pyridinic Ns, 1°/2°amino Ns and pyrrolic Ns in o-CDs.³⁸ The O_1s spectra (Fig. 5d) exhibited two peaks at 531.7 eV and 533.0 eV, respectively, which are attributed to C=O and C–OH/C–O–C bands in o-CDs. The results are in well agreement with the FITR analysis. The presence of such functional group is responsible for some properties of the as prepared o-CDs, such as water solubility and luminescence property.^36,46

Fig. 5 (a) XPS spectra; (b) XPS C1s spectra; (c) XPS O1s spectra; (d) XPS N1s spectra.

Mass spectra are able to map different types of chemical compound of o-CDs.⁶ Fig. 6a represents that there are some molecular skeletons formed by polymerides of o-PD, such as dipolymer proven by the m/z 212.1, tripolymer proven by m/z 316.2, tetramer proven by m/z 420.3, octamer proven by m/z 854.9, and the other fractured molecules proven by the other peaks of m/z, the dates of which did not marked, thus we can infer the formation process of o-CDs from o-PD. The o-CDs are gradually formed by the breaking of N–H bonds and C–H bonds of o-PDs and re-formation of N=C/N–C bonds, being accompanied by O–H, C–O, C=O, COO, between o-PDs sequentially under the condition of microwave radiation, which was described mainly in Fig. 6b. In addition, during the microwave heating, oxygen atoms existing in the solvent of H₂O and air on high temperature condition will also take part in the synthetic reaction of o-CDs. Therefore, we can speculate the synthesis of o-CDs is a process of micromolecule polymerizing into large molecules polymer containing plentiful C elements and a lot of unsaturated bonds.³⁸

Fig. 6 (a) Mass spectra of the as-prepared o-CDs, (b) the polymerization of o-phenylenediamine under the condition of microwave heating to form o-CDs.

Inspired by these prominent properties, such as strong PL and plentiful conjugated unsaturated groups on the surface of the as-prepared o-CDs, a kind of fluorescent sensing probe was designed to detect metal ions, pH values and H₂O₂ using the o-CD. We carry out the detection experiments by referring to the determination method of previous reports.^5,47–49 By detecting 9 kinds of metal ions, the concentrations of which were all 5 mM in the same way, and the results indicate that Fe²⁺, Fe³⁺ and Cu²⁺ showed the high quenching ability on the PL intensity (Fig. 7a and b). We further test the limit of detection (LOD) of Fe³⁺. Fig. 7c showed the F₀/F − 1 values of the as-prepared o-CDs solution treated with a certain concentration gradient of Fe³⁺. A good linear correlation (R² = 0.999) was exhibited over the concentration range of 0–100 nM and the LOD, according to the IUPAC standard, which was taken as 3× standard deviation/slope, was calculated as 16.1 nM.⁴³ The comparison of LOD of Fe³⁺ and H₂O₂ for different nano materials has been listed in Table 2. The detecting limit of o-CDs to Fe³⁺ is also much lower than what's reported in the previous reports.^16,48 The quenching phenomenon can be attributed to the nonspecific interaction of the complexation between Fe³⁺ and oxhydryl and carboxyl on the surface of the o-CDs,^5,50 and there may be electron transfer between the oxhydryl and carboxyl and Fe³⁺.^48,49

Fig. 7 (a) Spectra of fluorescence quenching upon addition of different metal ions; (b) the ratio of fluorescence intensity between the blank and solutions containing different metal ions; (c) sensitivity of the as-prepared o-CDs aqueous solution toward Fe³⁺ with a certain concentration gradient from 0 to 5 mM.

Table 2 Comparison of the PL detection of Fe³⁺ ions and H₂O₂ using different nano materials

Nano material	Synthesis method	LOD (nM)		Ref.
		Fe^3+	H2O2
a N-Doped CDs: N-doped carbon dots.b CDs: carbon dots.c S-Doped CDs: S-doped carbon dots.d GQD: graphene quantum dots.e Thiol-capped CdTeQD: thiol-capped CdTe quantum dots.
N-Doped CDs^a	Microwave	20		5
CDs^b	Microwave irradiation	211		16
S-Doped CDs^c	Hydrothermal	100		49
GQDs^d	Photo-Fenton reaction		700	51
Thiol-capped CdTe QD^e	Water phase synthesis		98	52
o-CDs	Microwave	16.1	28.1	This work

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Meanwhile, the as-prepared o-CDs were also used for pH value detection. As shown in Fig. 8a, there are fluorescence quenching phenomena with various pH.^47,53 When the pH value is changed from pH = 2 to 4, there are strongly fluorescence quenching. However, the strongest PL intensity of pH values is pH = 9 as shown in Fig. 8b. Additionally, there are obvious fluorescence quenching when the pH values are pH = 11 and 12. The results demonstrate that a strong acid or a strong alkali environment will both quench the fluorescence of o-CDs. The protonation and deprotonation of the carboxyl groups of the surface o-CDs cause electrostatic doping/charging of the o-CDs and shift the Fermi level, which cause the intensity changes of o-CDs.^54–56 Therefore, the water solution of the as-prepared o-CDs also has sensitivity to pH values.

Fig. 8 (a) Sensitivity of the as-prepared o-CDs aqueous solution toward different pH values; (b) the intensity of o-CDs solution mixed with phosphate buffers with different pH values.

Moreover, the sensing for H₂O₂ was also explored. Fig. 9 showed the F₀/F − 1 values of the as-prepared o-CDs solution treated with a certain concentration gradient of H₂O₂ aqueous solution, and its detecting limit is 28.1 nM, indicating water solution of the as-prepared o-CDs is sensitive to H₂O₂. The detecting limit of o-CDs is much lower than what's reported in previous publications.^51,52 The quenching phenomenon may be attributed to the redox reaction in the mixed solution of the o-CDs solution and H₂O₂, which is able to provide O₂ being apt to receive the e⁻ during a reaction, and there are many active groups providing the e⁻ on the surface of the as-prepared o-CDs, such as –COOH and –OH.^35,52,57,58 Therefore, the as-prepared o-CDs are able to be used to detect H₂O₂. Consequently, the as prepared o-CDs are able to be a kind of promising multifunctional probe for sensing Fe³⁺ ion, pH values and H₂O₂.

Fig. 9 Sensitivity of the as-prepared o-CDs aqueous solution toward H₂O₂ aqueous solution with a certain concentration gradient from 0 to 50 μM.

Meanwhile, the fluorescence lifetime of the o-CDs aqueous solution mixed with Fe³⁺ (5 mM), phosphate buffers (pH = 2), H₂O₂ (50 μM) and ultrapure water (blank control) at a ratio of 1 : 9 respectively was shown in Fig. 10. From Fig. 10, we can see that the fluorescence decay profiles are different. The fluorescence lifetime of o-CDs aqueous solution mixed with Fe³⁺ (5 mM), phosphate buffers (pH = 2), H₂O₂ (50 μM) and ultrapure water is 1.37, 0.008, 0.328 and 1.63 ns, respectively. The results indicate that the fluorescence lifetime of o-CDs aqueous solution mixed with Fe³⁺, phosphate buffers and H₂O₂ are lower than that of the counterpart with ultrapure water, which is able to demonstrate the occurrences of fluorescence quenching phenomenon.¹⁵

Fig. 10 PL lifetime decay profile of the aqueous solution of o-CDs aqueous solution mixed with Fe³⁺ (o-CDs + Fe³⁺), phosphate buffers (o-CDs + pH = 2), H₂O₂ (o-CDs + H₂O₂) and ultrapure water (o-CDs).

To evaluate the biocompatibility of the as-prepared o-CDs as imaging probes, we tested the cytotoxicity of the as-prepared o-CDs to SW480 cells. The cell viabilities of SW480 are all ≥90% when the concentrations of the as-prepared o-CDs were ≤1000 μg ml⁻¹ (shown in Fig. 11), suggesting low cytotoxicity in vitro and a good biocompatibility.

Fig. 11 Cell viability of SW480 cells after 24 h treatment with the as-prepared o-CDs of a concentration gradient of 0 μg ml⁻¹ to 1000 μg ml⁻¹ from CCK8 assay.

Furthermore, we studied the as-prepared o-CDs as a kind of fluorescent probe in vitro. While no obvious fluorescence of SW480 cells in the control groups was observed (shown in Fig. 12a–c), SW480 cells incubated with o-CDs (shown in Fig. 12d and e) can emit a green fluorescence clearly when they were exposed at a blue excitation light, indicating that the as-prepared o-CDs are able to be used as a kind of fluorescent probe for cell imaging, and have large potential applications as diagnostic reagent.

Fig. 12 (a–c) Fluorescence images of SW480 cells incubated without o-CDs under bright light, blue light and the overlay of (a) and (b), (d–f) fluorescence images of SW480 cells incubated with o-CDs under bright light, blue light and the overlay of (d) and (e). Scale bar = 20 μm.

4. Conclusion

The yellow fluorescence and water-soluble o-CDs with a high QY of 38.5% were synthesized by a one-step microwave-assisted method using o-phenylenediamine (oPD) as the precursor. o-CDs can emit strongly yellow fluorescence under the 365 nm UV light. Fe³⁺, different pH values and H₂O₂ are all able to quench the fluorescence of the water solution of the o-CDs at a certain degree and the as-prepared o-CDs can be developed as a kind of sensing probe for detecting Fe³⁺, pH values and H₂O₂. The detecting limit for Fe³⁺ (16.1 nM) and H₂O₂ (28.1 nM) is much lower than what's reported in previous studies. Moreover, fluorescence imaging experiment of SW480 cells demonstrates that the as-prepared o-CDs can be efficiently uptaken by cancer cells, revealing a quite good imaging effect. Therefore, the as-prepared yellow fluorescent o-CDs can be served as a promising multifunctional fluorescent probe for cell imaging and detecting Fe³⁺, pH and H₂O₂.

Acknowledgements

This work was supported by the State Key Project of Fundamental Research (Grants 2014CB931900 and 2012CB932504) of Ministry of Science and Technology of the People's Republic of China, the Chinese Academy of Sciences (“Hundred Talents Project”) and China Postdoctoral Science Foundation (119103S240).

References

1. R.-J. Fan, Q. Sun, L. Zhang, Y. Zhang and A.-H. Lu, Carbon, 2014, 71, 87–93.
2. W. Li, Z. Yue, C. Wang, W. Zhang and G. Liu, RSC Adv., 2013, 3, 20662–20665.
3. S. Y. Lim, W. Shen and Z. Gao, Chem. Soc. Rev., 2015, 44, 362–381.
4. M. Massey, M. Wu, E. M. Conroy and W. R. Algar, Curr. Opin. Biotechnol., 2014, 34, 30–40.
5. A. Zhao, C. Zhao, M. Li, J. Ren and X. Qu, Anal. Chim. Acta, 2014, 809, 128–133.
6. P. Miao, K. Han, Y. Tang, B. Wang, T. Lin and W. Cheng, Nanoscale, 2015, 7, 1586–1595.
7. X. Feng, Y. Zhao, L. Yan, Y. Zhang, Y. He, Y. Yang and X. Liu, J. Electron. Mater., 2015, 44, 3436–3443.
8. Q. Luo, Z. Wu, J. He, Y. Cao, W. A. Bhutto, W. Wang, X. Zheng, S. Li, S. Lin, L. Kong and J. Kang, Nanoscale Res. Lett., 2015, 10, 181–189.
9. M. Eita, A. Usman, A. O. El-Ballouli, E. Alarousu, O. M. Bakr and O. F. Mohammed, Small, 2015, 11, 112–118.
10. J. C. G. Esteves da Silva and H. M. R. Gonçalves, TrAC, Trends Anal. Chem., 2011, 30, 1327–1336.
11. V. Strauss, J. T. Margraf, C. Dolle, B. Butz, T. J. Nacken, J. Walter, W. Bauer, W. Peukert, E. Spiecker, T. Clark and D. M. Guldi, J. Am. Chem. Soc., 2014, 136, 17308–17316.
12. X. Yang, Y. Zhuo, S. Zhu, Y. Luo, Y. Feng and Y. Dou, Biosens. Bioelectron., 2014, 60, 292–298.
13. M. Algarra, M. Perez-Martin, M. Cifuentes-Rueda, J. Jimenez-Jimenez, J. C. Esteves da Silva, T. J. Bandosz, E. Rodriguez-Castellon, J. T. Lopez Navarrete and J. Casado, Nanoscale, 2014, 6, 9071–9077.
14. A. Qureshi, W. P. Kang, J. L. Davidson and Y. Gurbuz, Diamond Relat. Mater., 2009, 18, 1401–1420.
15. S. Chandra, S. Pradhan, S. Mitra, P. Patra, A. Bhattacharya, P. Pramanik and A. Goswami, Nanoscale, 2014, 6, 3647–3655.
16. R. Vikneswaran, S. Ramesh and R. Yahya, Mater. Lett., 2014, 136, 179–182.
17. J. Gu, D. Hu, W. Wang, Q. Zhang, Z. Meng, X. Jia and K. Xi, Biosens. Bioelectron., 2015, 68, 27–33.
18. J. Wang, Y. H. Ng, Y.-F. Lim and G. W. Ho, RSC Adv., 2014, 4, 44117–44123.
19. Z. Ma, H. Ming, H. Huang, Y. Liu and Z. Kang, New J. Chem., 2012, 36, 861–864.
20. S. Mohapatra, S. Sahu, N. Sinha and S. K. Bhutia, Analyst, 2015, 140, 1221–1228.
21. L. E. Santos-Figueroa, M. E. Moragues, E. Climent, A. Agostini, R. Martinez-Manez and F. Sancenon, Chem. Soc. Rev., 2013, 42, 3489–3613.
22. P. Pierrat, R. Wang, D. Kereselidze, M. Lux, P. Didier, A. Kichler, F. Pons and L. Lebeau, Biomaterials, 2015, 51, 290–302.
23. K. König, J. Microsc., 2000, 200, 83–104.
24. S. Zeng, Z. Yi, W. Lu, C. Qian, H. Wang, L. Rao, T. Zeng, H. Liu, H. Liu, B. Fei and J. Hao, Adv. Funct. Mater., 2014, 24, 4196–4196.
25. G. Tian, Z. Gu, L. Zhou, W. Yin, X. Liu, L. Yan, S. Jin, W. Ren, G. Xing, S. Li and Y. Zhao, Adv. Mater., 2012, 24, 1226–1231.
26. J. Stejskal, Prog. Polym. Sci., 2015, 41, 1–31.
27. K. Jiang, S. Sun, L. Zhang, Y. Lu, A. Wu, C. Cai and H. Lin, Angew. Chem., Int. Ed., 2015, 54, 5360–5363.
28. Q. Wang, X. Liu, L. Zhang and Y. Lv, Analyst, 2012, 137, 5392–5397.
29. R. Rajendran, S. Sohila, R. Muralidharan, C. Muthamizhchelvan and S. Ponnusamy, Eur. J. Inorg. Chem., 2014, 2014, 392–396.
30. Y. Liu, N. Xiao, N. Gong, H. Wang, X. Shi, W. Gu and L. Ye, Carbon, 2014, 68, 258–264.
31. C. Zhang, Z. Hu, L. Song, Y. Cui and X. Liu, New J. Chem., 2015, 39, 6201–6206.
32. B. Shi, L. Zhang, C. Lan, J. Zhao, Y. Su and S. Zhao, Talanta, 2015, 142, 131–139.
33. S. Capel-Cuevas, M. P. Cuellar, I. de Orbe-Paya, M. C. Pegalajar and L. F. Capitan-Vallvey, Anal. Chim. Acta, 2010, 681, 71–81.
34. B. Tang, F. Yu, P. Li, L. Tong, X. Duan, T. Xie and X. Wang, J. Am. Chem. Soc., 2009, 131, 3016–3023.
35. G. Wu, F. Zeng, C. Yu, S. Wu and W. Li, J. Mater. Chem. B, 2014, 2, 8528–8537.
36. C. Fu, L. Qiang, T. Liu, L. Tan, H. Shi, X. Chen, X. Ren and X. Meng, J. Mater. Chem. B, 2014, 2, 6978–6983.
37. L. Wang, Y. Wang, T. Xu, H. Liao, C. Yao, Y. Liu, Z. Li, Z. Chen, D. Pan, L. Sun and M. Wu, Nat. Commun., 2014, 5, 5357.
38. M. Vedamalai, A. P. Periasamy, C. W. Wang, Y. T. Tseng, L. C. Ho, C. C. Shih and H. T. Chang, Nanoscale, 2014, 6, 13119–13125.
39. L. Yang, Z. Li, G. Nie, Z. Zhang, X. Lu and C. Wang, Appl. Surf. Sci., 2014, 307, 601–607.
40. T. Zhang, S. Yang, J. Sun, X. Li, L. He, S. Yan, X. Kang, C. Hu and F. Liao, Synth. Met., 2013, 181, 86–91.
41. S. Yan, S. Yang, L. He, C. Ye, X. Song and F. Liao, Synth. Met., 2014, 198, 142–149.
42. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Springer, 2006.
43. X. Li, S. Zhang, S. A. Kulinich, Y. Liu and H. Zeng, Sci. Rep., 2014, 4, 1–8.
44. L. Bao, C. Liu, Z. L. Zhang and D. W. Pang, Adv. Mater., 2015, 27, 1663–1667.
45. J. Niu, H. Gao, L. Wang, S. Xin, G. Zhang, Q. Wang, L. Guo, W. Liu, X. Gao and Y. Wang, New J. Chem., 2014, 38, 1522–1527.
46. D. Tan, S. Zhou, Y. Shimotsuma, K. Miura and J. Qiu, Opt. Mater. Express, 2014, 4, 213–229.
47. H. Zhang, Y. Chen, M. Liang, L. Xu, S. Qi, H. Chen and X. Chen, Anal. Chem., 2014, 86, 9846–9852.
48. Q. Xu, P. Pu, J. Zhao, C. Dong, C. Gao, Y. Chen, J. Chen, Y. Liu and H. Zhou, J. Mater. Chem. A, 2015, 3, 542–546.
49. S. Liu, X. Qin, J. Tian, L. Wang and X. Sun, Sens. Actuators, B, 2012, 171–172, 886–890.
50. S. K. Sahoo, D. Sharma, R. K. Bera, G. Crisponi and J. F. Callan, Chem. Soc. Rev., 2012, 41, 7195–7227.
51. Y. Zhang, C. Wu, X. Zhou, X. Wu, Y. Yang, H. Wu, S. Guo and J. Zhang, Nanoscale, 2013, 5, 1816–1819.
52. Z. Wang, H. Song, H. Zhao and Y. Lv, Luminescence, 2013, 28, 259–264.
53. F. Y. Bo Tang, P. Li, L. Tong, X. Duan, T. Xie and X. Wang, J. Am. Chem. Soc., 2009, 131, 3016–3023.
54. H. Nie, M. Li, Q. Li, S. Liang, Y. Tan, L. Sheng, W. Shi and S. X.-A. Zhang, Chem. Mater., 2014, 26, 3104–3112.
55. S. Gomez-de Pedro, A. Salinas-Castillo, M. Ariza-Avidad, A. Lapresta-Fernandez, C. Sanchez-Gonzalez, C. S. Martinez-Cisneros, M. Puyol, L. F. Capitan-Vallvey and J. Alonso-Chamarro, Nanoscale, 2014, 6, 6018–6024.
56. S. Islam, N. Bidin, S. Riaz, G. Krishnan and S. Naseem, Sens. Actuators, A, 2016, 238, 8–18.
57. J. Wei, L. Qiang, J. Ren, X. Ren, F. Tang and X. Meng, Anal. Methods, 2014, 6, 1922–1927.
58. S. Chen, X. Hai, X. W. Chen and J. H. Wang, Anal. Chem., 2014, 86, 6689–6694.

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