Carbon Nanodots as Dual-Mode Nanosensors for Selective Detection of Hydrogen Peroxide
© The Author(s). 2017
Received: 26 March 2017
Accepted: 26 June 2017
Published: 6 July 2017
Hydrogen peroxide (H2O2) is an important product of oxidase-based enzymatic reactions, such as glucose/glucose oxidase (GOD) reaction. Therefore, the probing of generated H2O2 for achieving the detection of various carbohydrates and their oxidases is very significative. Herein, we report one kind of dual-emission carbon nanodots (CDs) that can serve as novel dual-mode nanosensors with both fluorometric and colorimetric output for the selective detection of H2O2. The dual-model nanosensors are established only by the undecorated dual-emission CDs, where significant fluorometric and colorimetric changes are observed with the addition of different concentrations of H2O2 in the CD solution, which benefit to the achievement of the naked-eye detection for H2O2. The mechanism of the nanosensors can be attributed to the fact that the external chemical stimuli like hydroxyl radicals from H2O2 bring about the change of surface properties and the aggregation of CDs, which dominate the emission and absorption of CDs. The constructed dual-mode nanosensors exhibit good biocompatibility and high selectivity toward H2O2 with a linear detection range spanning from 0.05 to 0.5 M and allow the detection of H2O2 as low as 14 mM.
KeywordsCarbon nanodots Fluorescence Dual-mode nanosensors Detection
Fluorescent carbon nanodots (CDs) have triggered extensive research attention for their unique physicochemical properties like good biocompatibility, low toxicity, tunable photoluminescence (PL), and high quantum yield. Because of the above characters, CDs have found potential applications in a variety of fields including but not limited to bioimaging, biosensors, and light-emitting devices [1–9]. Moreover, due to their up-conversion and down-conversion ability, lack of optical blinking, and high photostability compared to organic dyes or semiconductor quantum dots (QDs), CDs are more suitable for applications in fluorescent nanosensors by fluorescence increase or quenching [10–19].
Hydrogen peroxide (H2O2) is one kind of common oxidizer, which is always used as medical disinfectant for the ability of sterilization. Besides, H2O2 is also an important product of oxidase-based enzymatic reactions, such as glucose/glucose oxidase (GOD) reaction. Therefore, the sensing strategy through the probing of H2O2 can be employed as a promising approach for the detection of carbohydrates and their oxidases. For this reason, the sensing of H2O2 may be used to monitor the diseases about carbohydrate metabolism, such as diabetes. Currently, although various glucose sensors based on the determination of H2O2 have been developed by using a variety of analytical methods, previously reported sensor systems are mainly based on a single signal such as conductometric, fluorometric, or colorimetric change [20–22]. Recently, advances in nanotechnology, especially in fluorescent nanoparticles like semiconductor QDs and emerging carbon-based nanoparticles have brought about novel H2O2 nanosensors. Lu et al. developed one kind of dual-emission microhybrids (DEMBs) by combining CdTe QDs and rhodamine for ratiometric fluorescent sensing of glucose through monitoring the generation of H2O2 . Zhang et al. reported a fluorescent nanosensor that showed selective and sensitive response to H2O2 through the fluorescence quenching of CDs [21, 22]. However, these work inevitably brought about the intrinsic defects of semiconductor-based QDs with expensive chemical constituents and heavy metal pollution. Moreover, the nanosensors based on single signal readout, either fluorescence quenching or color change, may have poor assay stability due to the fluctuations of environmental factors and the experimental operation errors. On account of the above consideration, we wish to develop a new class of fluorescent CDs, whose fluorescence and solution color are very sensitive to the change of the concentrations of H2O2. Thus, a dual-mode nanosensor based on these CDs can be achieved for distinctively and sensitively sensing the H2O2 by simultaneously inspecting the fluorometric and colorimetric changes of CD solution, which is beneficial to the realization of naked-eye detection of the H2O2.
In this study, we have developed a facile and convenient method to synthesize a novel type of CDs, which exhibits a dark red solution color under visible light and dual fluorescent emission under a 365-nm UV lamp (blue and green fluorescence emission). The CDs are simply synthesized through solvothermal method with citric acid, urea, and N,N-dimethylformamide (DMF) as carbon source, nitrogen source, and reaction solvent, respectively. The fluorescence and solution color are very sensitive to changes in the concentrations of H2O2. Thus, a dual-mode nanosensor based on these CDs can be achieved for distinctively and sensitively sensing the H2O2 by simultaneously inspecting the fluorometric and colorimetric changes of the CD solution, which is beneficial to the realization of naked-eye detection of the H2O2. Without the introduction of any expensive instrument, a dual-mode nanosensor based on these CDs has been established. This sensing system may effectively avoid the potential operation errors and markedly improve the reliability of the measurement. In addition, the CD-based nanosensors are promising in the application of blood glucose detection both in vivo and in vitro owing to their good biocompatibility and high water solubility.
Synthesis of CDs
The CDs were prepared using a solvothermal method with citric acid as the carbon source, urea as the nitrogen source, and DMF as the co-reactant. In a typical experiment, citric acid (1 g) and urea (2 g) were dissolved in 10 mL DMF. The solution was then transferred to a 25-mL poly(tetrafluoroethylene)-lined autoclave and heated at 160 °C for 4 h. After the reaction, the autoclave was naturally cooled down to room temperature. A dark red solution was obtained. The CDs were precipitated by adding 5 mL reaction solution into 25 mL abundant ethanol and centrifuged at 7500 rpm for 30 min. Then, the precipitation was dialyzed to obtain pure CDs. The as-prepared CDs were collected and dried in a vacuum drying oven at 60 °C and under <1 Pa for 12 h. Then, the CDs were redissolved in deionized water to form 0.75 mg mL−1 CD solution for further research. And the subsequent H2O2-treated CDs were collected and dried with the same method for the characterization of the surface morphology and structural properties.
The surface morphology of the CDs was characterized by a high-resolution transmission electron microscope (HRTEM, JEOL JSM-IT100). The structural properties of the CDs were performed by an X-ray diffractometer (XRD, PA National X’Pert Pro) and a micro-Raman spectrometer (Renishaw RM 2000). The absorption spectra of the CDs were measured on a Hitachi U-3900 UV-Vis-NIR spectrophotometer. The fluorescence spectra of the CDs were measured by a spectrophotometer (Hitachi F-7000). The fluorescence quantum yield of the CDs was obtained by the Horiba FL-322 spectrometer with a calibrated integrating sphere. The fluorescence decay curves of the CDs were also measured by Horiba FL-322 using a 405-nm NanoLED monitoring the emission at 450 and 500 nm, respectively. The Fourier transform infrared spectrum (FTIR) of the CDs was recorded on a Bio-Rad Excalibur spectrometer (Bruker Vector 22). X-ray photoelectron spectroscopy (XPS) was recorded on an ESCALAB MK II X-ray photoelectron spectrometer using Mg as the exciting source.
Establishment of the CD Nanosensors
For the detection of the H2O2, the fluorescence and absorption spectra of the CDs in the presence of H2O2 were examined in PBS buffer (pH = 7.4, at 25 °C). In a typical experiment, a different amount of H2O2 was mixed with distilled water firstly and then 20 μL 0.75 mg mL−1 CD solution was injected into 4 mL H2O2 solution with different concentrations (0, 0.05, 0.1, 0.15, 0.25, 0.5, 1.0, and 2.0 M). Then, photographs, fluorescence, and absorption spectra were taken after the CDs were added into the H2O2 solution.
The selectivity of the CD-based nanosensors was also evaluated. The CD solution (20 μL, 3.75 μg mL−1) was mixed with different kinds of cations and oxidants (4 mL, 0.1 M) and then the solution was shaken for 1 min. At last, the UV-Vis absorption and fluorescence spectra of the solution were recorded after the CDs were added into the H2O2 solution.
Results and Discussion
Characterization of the CDs
The fluorescent behavior of the CD-based nanosensors toward H2O2 was measured in the CD aqueous solutions shown in Fig. 1e. Under a single wavelength excitation at 365 nm, the CD solutions illustrate asymmetrical emission spectra, which could be fitted by dual-emission fluorescent bands centered at 450 and 500 nm, corresponding to blue and green fluorescent bands, respectively. When the CD solutions are mixed with H2O2, the intensity of the blue band demonstrates a greater decrease than that of the green one. Accordingly, the strongest emissions of the CDs shift from 450 to 500 nm from the results of the excitation-emission matrices of the CDs after the addition of the H2O2 (Additional file 1: Figure S2). As a result, the fluorescence color of the CD solutions changes from blue to green under a 365-nm UV lamp illumination (inset of the Fig. 1e). Moreover, the CD solutions simultaneously experience a colorimetric change from dark red to green after adding the H2O2 (inset of the Fig. 1f). This color change can be attributed to the intensity evolution of the absorption bands at around 555 and 595 nm caused by the addition of H2O2 in the CD solution (Fig. 1f). Taken together, these results confirm the CDs could be used as a dual-mode nanosensor for the detection of H2O2.
To investigate the sensing mechanism, the morphology and fluorescence properties of the CDs after adding H2O2 were also characterized. As illustrated in Additional file 1: Figures S1a and S1c, the addition of H2O2 into the CD solution can lead to the aggregation of CDs, whose sizes are ranging from 30 to 60 nm. The H2O2-induced aggregation of CDs was also revealed in the normalized absorption spectra (Additional file 1: Figure S3); namely, the absorption band of the CDs red-shifts from 555 to 595 nm in the visible region . Correspondingly, the color of the CD solution varies from dark red to green, along with the dispersion state of CDs turning into an aggregation state. The XRD spectra (Fig. 1b and Additional file 1: Figure S4) of the CDs before and after adding H2O2 alter little, indicating there are no changes in the crystalline structure of the CDs.
Photophysical data for CDs (3.75 μg mL−1 in deionized water) before and after 0.5 M H2O2 treatment
λ em a (nm)
τ b (ns)
The FTIR and XPS spectra of the CDs were measured to give insight into the chemical composition and environmental changes caused by the H2O2. The FTIR spectra of CDs before and after adding H2O2 shown in Additional file 1: Figure S7 illustrate that the stretching vibrations of N–O at around 1350–1390 cm−1 increase with the addition of H2O2, which is also confirmed by the result of the XPS spectra. It is observed from the full survey XPS spectra (Additional file 1: Figure S8) that the O to N ratio of the CDs before and after the H2O2 treatment was 1.57 and 3.85, respectively. The increasing ratio of O/N reveals that the bonding states of N in the CDs may change with the addition of the H2O2, which is in line with the high-resolution N1s XPS spectra shown in Fig. 2c, d. From the result of the N1s XPS spectra, the content of graphite N in the CDs has been increased with the addition of the H2O2. Furthermore, there is an additional peak of N–O state at 407.3 eV in the N1s spectra after the addition of the H2O2, which obviously demonstrates that the importing of the H2O2 brings about the variation of the surface states in the CDs. All the surveys manifest that the surface N frame could be changed by the addition of the H2O2.
Evaluation of the CDs Nanosensors
On the basis of the above fluorescent and colorimetric behavior of the CDs, we have developed a nanosensor to detect H2O2 by the CDs. The proposed sensing system is consisted of CDs with proper concentration in aqueous solution (3.75 μg mL−1, Additional file 1: Figure S9), where the CDs serve dual function as both colorimetric and fluorometric reporters in this system.
In conclusion, we propose a dual-mode nanosensor based on CDs with both colorimetric and fluorometric output for the quantitative detection of H2O2 based on the fluorometric and colorimetric change of the CD solution upon the introduction of H2O2. The nanosensors are simple and facile to achieve naked-eye detection for H2O2. The mechanism of the nanosensors can be attributed to the fact that the external chemical stimuli like hydroxyl radicals from H2O2 bring about the change of surface properties and the aggregation of CDs, which dominate the emission and absorption of CDs. The proposed nanosensors exhibit good biocompatibility, high selectivity toward H2O2 with a linear detection range spanning from 0.05 to 0.5 M, and a detection limit of around 14 mM, which is comparable to the level of H2O2 produced by the GOD reactions. It is believed that the strategy reported in this paper may provide a promising approach for developing a novel sensor in blood glucose, which could be valuable in disease diagnosis and environmental testing.
This work is financially supported by the National Science Foundation for Distinguished Young Scholars of China (61425021); the National Natural Science Foundation of China (51602288, 11374296, and 51272238); China Postdoctoral Science Foundation (2015M582192 and 2016T90671); and Startup Research Fund of Zhengzhou University (1512317006).
CXS conceived the idea and supervised the project. CLS and QL designed and conducted the experiments. CLS, LXS, QL, CXS, and JHZ performed the data analysis. CLS, QL, JHZ, CXS, and XJL wrote the manuscript. All of the authors discussed the manuscript. All the authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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