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In this paper, the multicolor photoluminescence carbon quantum dots (CQDs) have been synthesized by one step hydrothermal method to analyze hydrogen peroxide (H2O2). These CQDs exhibited multicolor emission under a single wavelength excitation for the quantification of H2O2. Using an LED with a central wavelength of 365 nm as the excitation source, it is shown that the red emission CQDs capable of detecting H2O2 over the linear range of 0-88.2 mM and the H2O2 sensitivity of wavelength shift to changes in the H2O2 concentration was found to be 0.18 nm/mM. These results of the optical sensing method can be used in practice detection of H2O2 and could offer a new approach for developing a new biosensor. The CQDs exhibit good emission property and high stability, as well as excitation-independent emission behavior. Moreover, it is attractive that CQDs can be used as an effective fluorescent probe for the detection of H2O2 with linear Stern-Volmer plot and wavelength shift in an aqueous solution.
© 2016 Optical Society of America
1. Introduction
Hydrogen peroxide (H2O2) plays an essential role as an oxidizing, bleaching and sterilizing agent in biochemical and chemical industry [1]. High concentrations of H2O2 would cause irritation to dyes and shin, it affects human health [2]. On the other hand, the determination of H2O2 concentration is important in food [3], clinical [4] and environmental applications [5]. There are many methods which can be used for the quantitative analysis of H2O2 in solution, such as electrochemical method [6–8] and spectroscopy [9–14]. The electrochemical methods are well suited determining lower concentrations of H2O2, however, they can suffer interference from other reactive oxygen species. Furthermore, electrochemical detection methods are all susceptible in the environment with significant electromagnetic interference. On the other hand, the field of spectroscopic detection method can be subdivided into chemiluminescence [9–12], and spectrofluorometry [13, 14]. The spectroscopic techniques can also be used for detection of H2O2 at low concentrations. The spectroscopic techniques have been integrated with luminescent quantum dots (QDs), making possible the development of optical sensors with high sensitivity and many of advantages of luminescent QDs, such as small size, high selectivity and immunity to electromagnetic interference.
In the last decade, there has been a focus on the luminescent carbon quantum dots (CQDs) to further improve the optical sensing technique due to their unique optical properties caused by the quantum confinement effect. The luminescent carbon quantum dots were first produced in 2006 [15]. Compare to semiconductor quantum dots, luminescent CQDs were considered as a brand new class of luminescent due to their small size, high quantum yield, low cytotoxicity, possess fine biocompatibility, high photostability and environment friendliness, which make them suitable chemical and biological analyses. There are several methods to prepare CQDs have been reported [16–20]. However, these methods usually require not only complicated equipment and expensive materials but also complicated post treatment to enhance the quantum yield and water solubility of CQDs. Meanwhile, the isolation, purification and functionalization processes based on these methods are all complicated and time consuming. Therefore, the hydrothermal method has been widely used to prepare various luminescent QDs because of the high reactivity of the reactants, easy control of the solution, low level of air pollution and low energy consumption under hydrothermal conditions [21].
In this work, we have presented a new and simple H2O2 sensing technique based on the fluorescence quenching and wavelength shift of CQDs. Fluorescence quenching refers to any process which decreases the fluorescence intensity of a certain fluorophore due to various molecular interactions. The optical sensing method was composed of CQDs without any complex processes of functionalization or conjugation. The obtained optical sensing method showed the linear detection range and wavelength shift. The sensitivity enables the use of the QDs as H2O2 sensors and provides a versatile fluorescent reporter for the activities of oxidases and for the detection of their substrates.
2. Experimental
The multicolor luminescence CQDs were synthesized from sodium citrate and NH4HCO3 through a simple, convenient and one-step hydrothermal method. Briefly, sodium citrate (0.3 g), NH4HCO3 (3.1 g) and water (20 mL) were sealed into a Teflon equipped stainless steel autoclave, which was then placed in a drying oven followed by hydrothermal treatment at 200 °C for 4 h. After the reaction, the autoclave was cooled to room temperature. The purification of the multicolor luminescence CQDs was conducted through a dialysis tube for about 24 h in dark. Based on the different rates of temperature increase, the blue, green and red emission of CQDs can be obtained.
Figures 1(a)-1(c) show the transmission electron microscopy (TEM) images of multicolor luminescence CQDs. These samples for TEM measurement were prepared by deposition of on drop of aqueous dispersion on a copper grid coated with CQDs and solvent was removed by evaporation in the oven at 60 °C. Figure 1(d) presents the energy-dispersive X-ray spectroscopy (EDX) results for the composition of the CQDs. The nanoparticles are composed principally of O, C, and Cu elements, where the x axis represents energy (keV) and the y axis represents the counts per second per electron (basically X-ray intensity). The Cu content originates from the copper grid, the C content originates from the CQDs.
Fig. 1 TEM images of (a) red emission (b) green emission and (c) blue emission CQDs (d) EDX analysis results for CQDs.
Figure 2 shows a schematic illustration of the experimental arrangement used to characterize the performance of the optical H2O2 sensing method. In the optical sensing experiments, the fluorescence excitation was provided by an LED (NSHU591B, NICHIA, COPR.) with a central wavelength of 365 nm driven by an arbitrary waveform generator (TGA1240, Thurlby Thandar Instruments (TTI) Ltd.) at 10 kHz in pulse signal mode. The emission measurements were acquired using a USB 4000 spectrometer (Ocean Optics).
3. Results and discussion
Figures 3(a)-3(c) present the room-temperature fluorescence spectra of the multicolor emission CQDs under different H2O2 concentrations. These CQDs exhibited red, green and blue emission under an LED with a central wavelength of 365 nm as the excitation source. Note that in acquiring the relative fluorescence intensity data, the integration time of the CCD spectrometer was set at 10 ms, and as a result, the red, green and blue emission CQDs exhibit strong fluorescent emissions at 604 nm, 550 nm and 445 nm, respectively. Figure 3 shows that the relative fluorescence intensity of the multicolor luminescence CQDs decreases significantly as the H2O2 concentration increases.
Fig. 3 Emission spectra of multicolor luminescence CQDs under different H2O2 concentrations: (a) red emission (b) green emission and (c) blue emission CQDs. Insert: photographs of each king of luminescence CQDs solution.
Figure 4(a) shows the variation of peak red emission wavelength with different H2O2 concentrations. It can be seen that the peak red emission wavelength increases as the H2O2 concentration increase. Figure 4(b) shows the H2O2 sensitivity of the wavelength shift to change in the H2O2 was found to be 0.18 nm/mM. The H2O2-dependent variation of fluorescence intensity at a peak 604 nm is replotted in the form also shown in Fig. 4(b). It can be seen that the fluorescence intensity reduces exponentially as the H2O2 concentration increased over the range of 0-88.2 mM. In other words, the fluorescence intensity is reduced as a result H2O2 quenching. From inspection, the exponential correlation coefficient is found to be R2 = 0.9898.
Fig. 4 (a) variation peak emission wavelength with H2O2 concentration and (b) variation of peak emission intensity and wavelength with H2O2 concentration.
It has been shown that both the fluorescence intensity and peak red emission wavelength of CQDs, have a linear dependence on H2O2 concentration in the environmental range. When the H2O2 is increased, fluorescence intensity decreases, due to increase of non-radiative transitions. The peak wavelength, on the other hand, is shifted towards longer wavelengths. Either effect can be used to obtain H2O2 concentration information. However, simple fluorescence intensity measurements are prone to error due to optical power fluctuations. Due to the presence of a wavelength shift is proportional to H2O2 concentration and independent of system optical power level.
In a homogeneous microenvironment, the emission intensity of the multicolor CQDs is expected to follow the quenching effect can be described by the Stern-Volmer equation [22]:
where I0 and I represent the steady-state fluorescence intensities in the absence and presence of H2O2, respectively; KSV is the Stern-Volmer quenching constant; [Q] represents the concentration of quencher (H2O2). For this ideal case, a plot of I0/I vs. [Q] will be linear with a slope equal to KSV and an intercept of unity.Figure 5 presents the Stern-Volmer plots for multicolor luminescence CQDs. A significant decrement of the fluorescence intensities of multicolor CQDs are observed as the concentration of H2O2 is increased. Stern-Volmer plots show linear relationship. H2O2 is a good quencher for the multicolor CQDs and from the Stern-Volmer plot, the concentration of H2O2 could be estimated.
Fig. 5 Stern-Volmer plots of multicolor luminescence CQDs: (◆) red emission, (■) green emission and (▲) blue emission CQDs.
The optical parameters for H2O2 sensing materials are summarized in Table 1. Extensive studies have been reported on absorbance-based [23–27] and fluorescence-based [28–32] H2O2 sensing method. The currently available optical sensing methods utilized the changes in absorbance or luminescence intensity as detecting signal. A major limitation of the intensity-based sensing method is that the signal output can be interfered by the factors such as excitation source intensity, environmental conditions, sensor distribution and instrumental efficiency. By contrast, a wavelength shift measurement in this study provides a more precise and sensitive evaluation of H2O2 concentration changes. In this work, multicolor luminescence CQDs were synthesized by one step hydrothermal method and their H2O2 sensing properties were studied. Results show that the red emission CQDs demonstrates capable of detecting H2O2 over the linear range of 0-88.2 mM and the H2O2 sensitivity of wavelength shift to changes in the H2O2 concentration was found to be 0.18 nm/mM.
Table 1. Comparison of performance characteristics of proposed optical H2O2 sensing method with those of existing optical H2O2 sensing method.
Figure 6(a) shows that the photostability of proposed optical H2O2 sensing method using multicolor luminescence CQDs. The photostability was also tested by placing the multicolor luminescence CQDs in cuvette with pulse irradiation with 365 nm wavelength LED at room temperature for around 3.5 h. After continuous illumination for around 3.5 h, the relative fluorescence intensities of multicolor luminescence CQDs are stable. On the other hand, the high power photostability experiment was also performed by using a 365 nm UV light source (UVP C-10, lamp power: 6 W) for up to 24 h. A plot of fluorescence intensity as a function of irradiation time is shown in Fig. 6(b). After continuous irradiation for around 6 h, the carbon quantum dots could be partially destroyed by high power UV light source. Approximately 18% decrease in fluorescence intensity was observed after 24 h of irradiation.
Fig. 6 (a) Photostability of multicolor luminescence CQDs (b) fluorescence intensity of CQDs solution as a function of irradiation time (365 nm UV light, lamp power: 6 W).
4. Conclusion
In this study, we synthesized water-soluble multicolor luminescence CQDs by one step hydrothermal method using a common carbon resource. The multicolor luminescence CQDs have been successfully applied to develop new fluorescent biosensors for sensitive detection of H2O2. Using an LED with a central wavelength of 365 nm as the excitation source, the red emission CQDs exhibited a linear range form 0-88.2 mM for the determination of H2O2 and the H2O2 sensitivity of wavelength shift to changes in the H2O2 concentration was found to be 0.18 nm/mM. The multicolor luminescence CQDs for H2O2 exhibit good analytical performance with low cost, convenient and sensitive detection. The multicolor luminescence CQDs may also be a promising candidate for drug delivery, biochemistry and biological applications.
Acknowledgments
The authors gratefully acknowledge the financial support provided to this study by the Ministry of Science and Technology of Taiwan under Grant No. MOST 104-2221-E-131-029.
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