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Facile green and one-pot synthesis of purple perilla derived carbon quantum dot as a fluorescent sensor for silver ion
Talanta 201 (2019) 1–8
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Facile green and one-pot synthesis of purple perilla derived carbon quantum dot as a fluorescent sensor for silver ion
T
Xinyi Zhaoa, Sen Liaoa, Lumin Wanga, Qi Liua,∗∗, Xiaoqing Chena,b,∗ a b
College of Chemistry and Chemical Engineering, Central South University, Changsha, 410083, Hunan, China Key Laboratory of Hunan Province for Water Environment and Agriculture Product Safety, Central South University, Changsha, 410083, Hunan, China
ABSTRACT
In this work, biomass-derived carbon quantum dots (CQDs) with excellent water solubility, strong fluorescence and favorable biocompatibility were synthesized via one-step hydrothermal treatment of purple perilla for the first time. The functional group composition, morphology, and pH stability of the synthesized CQDs were systematically investigated. And based on fluorescence quenching of CQDs, the as-prepared CQDs were innovatively developed as an effective “signal-off” fluorescent probe for selective and sensitive detection of silver ion (Ag+) with two linear ranges of 0–10 and 10–3000 nM, and a detection limit 1.4 nM. The specificity and selectivity of this fluorescent probe were also verified through challenging the detection by using similarmetallic cations or in real water samples. In addition, the asprepared CQDs exhibit a low cytotoxicity and a good biocompatibility, revealing its potential bioimaging applications in living cells.
1. Introduction Resulting from its advantages including high quantum yield, well water solubility, excellent chemical stability, good biocompatibility and ease of modification, carbon quantum dots (CQDs) have recently emerged as a new nanocarbon materials used for bioimaging, biosensing, drug delivery, efficient visible light-active photocatalysts and so forth [1–7]. Generally, the synthetic method to produce CQDs can be divided into two broad categories, namely bottom-up and top-down. And production of CQDs can be realized through synthetic approaches such as arc-discharge, laser ablation, electrochemical, hydrothermal, ultrasonic and microwave treatment [8–12]. However, the majority of above approaches have some limitations, such as limited spectral efficiency, low product yield, lack of size control, and the use of toxic chemicals or high temperature for experiments. Hydrothermal carbonization has arisen as a powerful and sustainable technology for the synthesis of CQDs in aqueous media since it is green, facile, convenient and efficient. After first biomass-derived CQDs obtained by hydrothermal treatment of grass in 2012 [13], more attentions have been paid to producing CQDs from natural plants, such as cabbage, sweet red pepper, aloe and various vegetables [14]. Especially, biomass-derived CQDs can be easily fabricated by using the variety of natural carbon sources. It also can effectively avoid the use of costly/toxic chemicals and complicated post-treatment processes. In addition, these CQDs derived from biomass have numerbers of advantages including cheap raw materials, easy contronl, massproduction and high yield. Moreover,
∗
bioprotein [14–16] and wastes (such as waste pololefins [17], fish scales [18] and even human urine [19]) also can be used as carbon sources for preparing CQDs. In summary, there are multiple inherent advantages in reusing the waste materials and natural bioresources, such as abundance of carbon sources from low-value precursor materials, varieties of heteroatom doping (such as N, S, P), saving of chemical precursors, addressing of environmental issues caused by waste disposal etc [17,20]. Perilla frutescens (L.) Britt. referred to as purple perilla (also called Zisu in China), belonging to the family Labiatae, is distributed worldwide, especially in China, Japan, Korea, and other regions in Asia [21]. In China, the plant of purple perilla is not only used as a traditionally medicinal herb for various diseases, but also as a common flavor for fish and crab cooking with the purposes of detoxification. Till now, various compounds including flavonoids, volatile oils, fatty acids, triterpenes, phenolic compounds have been isolated and identified from this plant, which can serve as an excellent source of carbon and nitrogen to produce CQDs. And compared with organic molecules as precursor to synthesize CQDs, the natural source of carbon and nitrogen from purple perilla possess great characteristics including convenient sources, low price, good water solubility, innocuity and no pollution to environment. Silver ion (Ag+) is one of several essential trace element species in the human body, and toxicity acts as a stimulant for the production of red blood cells. Nevertheless, high level of Ag+ in organisms might cause serious consequences. And the potential toxicity is continuously escalated as the Ag+ being accumulated by aquatic organisms to poison
Corresponding author. College of Chemistry and Chemical Engineering, Central South University, Changsha, 410083, Hunan, China. Corresponding author. College of Chemistry and Chemical Engineering, Central South University, Changsha, 410083, Hunan, China. E-mail addresses: [email protected] (Q. Liu), [email protected] (X. Chen).
∗∗
https://doi.org/10.1016/j.talanta.2019.03.095 Received 30 September 2018; Received in revised form 22 March 2019; Accepted 27 March 2019 Available online 30 March 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
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Scheme 1. Illustration of the synthetic procedure of the CQDs and the CQDs-based fluorescence biosensing platform for the detection of Ag+.
aquatic animals, plants, and even human body in previously reports [22]. Due to its extreme toxicity, the maximum permissive level of Ag+ in drinking water is set at 0.05 ppm by the World Health Organization [23]. Consequently, monitoring the levels of Ag+ in the environment is deemed important for environmental protection and health reasons. Till now, methods such as atomic absorption spectroscopy [24], electrochemistry [25], fluorescence, and inductively coupled plasma mass spectroscopy have been used to detect metal ions. In which, the fluorescence analysis method is currently considered to be a highly effective way for Ag+ detection. Therefore, it is of great significance to design and prepare a highly sensitive and selective fluorescent probe for Ag+ detection. Compared with other fluorescent materials (conventional dyes, polymers, and semiconductor quantum dots), CQDs could offer the advantageous features of bright fluorescence, high photostability, low toxicity, and resistance to metabolic degradation in bio-applications [26]. In the present study, purple perilla was selected as the source for the facile one-pot synthesis of biomass-derived CQDs for the first time. And, as shown in Scheme 1, the resulting blue fluorescent CQDs was then served as the probe fordetection of silver ion because the luminescence can be effectively quenched by the target (Ag+). The specificity and selectivity of the CQDs probe were also evaluated via challenging the detection by using other metal cations or in complicated lake water. Besides, the related mechanisms of the CQDs formation and the Ag+-induced fluorescence quenching were also illustrated.
2600 UV–Vis spectrophotometer (Shimadzu, Japan) was introduced for UV–vis absorption measurements. The Zeta potentials were measured on a Zetasizer Nano ZS (Malvern Instruments Ltd., United Kingdom) equipped with a noninvasive back scattering (NIBS) device and folded capillary sample cell (for Zeta potentials). 2.3. Synthesis of CQDs CQDs were synthesized via a one-step hydrothermal treatment of purple perilla in Teflon lined autoclave. Initially, dried purple perilla was purchased from a local Herbalists and smashed by a domestic pulverizer. And the moderate power of purple perilla was added to 30 mL of distilled water in a Teflon lined hydrothermal reactor. Then the solution was transferred into a 50 mL poly(tetrafluoroethylene)-lined auto-clave after the ultrasonic treatment for a period of time, and heated at 260 °C for 5 h. After being cooled down to room temperature, the light brown suspension was collected through a 0.22 μm micro-pore film filter to eliminate the unreacted raw materials and the large dots. Finally, the obtained CQDs were dialyzed against ultrapure water through a dialysis membrane (MWAG of 100 Da) for 2 days to remove impurities while ultrapure water was changed with an interval of 8 h. Then the dispersion was stored at 4 °C. 2.4. Detection of Ag+
2. Experimental
CQDs was adjusted to a concentration of 300 μg mL−1 in 0.1 mM aqueous solutions containing twenty-five different ions (including Ag+, Co2+, Mg2+, Hg2+, Ni2+, Sr2+, Ca2+, Pb2+, Li+, Zn2+, Cu2+, Cd2+, Fe3+, Fe2+, Cr3+, Al3+, Mn2+, Na+, K+, SO42−, Cl−, S2−, NO3−, CO32−, SO32−), and then subjected it to fluorescence measurements after 15 min. The quenching effect of Ag+ on fluorescence intensity of CQDs was conducted as follows. Typically, 1 mL of CQDs aqueous solution (300 μg mL−1 in deionized water, pH 7.0) was placed in each of 4.0 mL EP tubes, and then the final aqueous systems were acquired by the addition of a calculated amount of Ag+ stock solution, respectively. The final concentrations of Ag+ presented were from 0.5 to 1.0 × 108 nM. The fluorescence emission intensity at 450 nm of the blank sample excited at 360 nm were measured and marked as F0. After equilibration for 15 min, the fluorescent intensity (F) of each sample was measured.
2.1. Materials and reagents Dried purple perilla was bought from local Herbalists (Changsha, China). Quinine sulfate was purchased from Aladdin (Shanghai, China). Na2SO4, Na2SO3, CoSO4, AgNO3, Cu(NO3)2, Pb(NO3)2, LiCl, Zn(NO3)2, Cd(NO3)2, NiSO4, MgSO4, CaCl2, SrCl2, HgCl2, FeCl3.6H2O, FeSO4, MnCl2, CrCl3, AlCl3, KCl, NaCl, Na2S, NaNO3 and Na2CO3 were supplied by Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). All reagents were analytical reagent grade. All aqueous solutions were diluted by ultrapure water with a resistivity≥ 18.2 MΩ cm−1 form a Molecular water purification system. 2.2. Instruments
2.5. Analysis of practical samples
Transmission electron microscope (TEM) images displaying the morphological evaluation and sizes of CQDs were analyzed by a HT7700 transmission electron microscope (TEM, Hitachi) under an accelerating voltage of 200 kV. The surface functional properties of CQDs were investigated by PHI Quantera Ⅱ X-ray photoelectron spectroscopy (XPS, Thermo Fisher Scientific 250Xi). Infrared spectra were obtained using IR Prestige-21 Fourier transform infrared (FTIR) (Shimadzu, Japan). The fluorescence and time-resolved fluorescence decay tests were conducted using a LS-55 spectrofluorometer (PerkinElmer, America) and a Fluo Time 100 compact fluorescence lifetime spectrophotometer (PicoQuant, Germany), respectively. A UV-
To evaluate the viability and practicality of the developed CQDs sensor for Ag+ detection in practical water samples, the performance of the current method was examined by lake water samples which obtained from Xiangjiang River of Changsha (China) and tap water samples from our lab. The obtained real water samples were filtered through 0.22 μm micro-pore film filter. The river water was added standard Ag+ solutions with different concentrations levels, which was analyzed by the proposed method. The tap water samples were spiked with the different concentrations of Ag+ solution directly without any pretreatment. 2
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2.6. Cytotoxicity assay and cellular imaging of CQDs
oxygen and nitrogen, respectively [29]. And there are four main peaks in the high-resolution spectrum of C 1s in Fig. 2(b), indicating the presence of four types of carbon bonds, corresponding to sp2 C (C−C or C=C) in graphene at 284.5 eV, sp3 C from C−N and C−O at 285.5 eV, C=N at 287.1 eV, and C=O at 288.7 eV [30,31]. Besides, as displayed in Fig. 2(c), there are two peaks 531.9 and 532.9 eV present in the O 1s spectrum, which can be assigned as the C=O and C–OH groups, respectively [32,33]. Additionally, deconvolution of N 1s spectra in Fig. 2(d) reveals two relative nitrogen species of N−H (400.1 eV) and C−N (401.4 eV) [34]. As for the FT-IR spectrum of CQDs (Fig. 3(a)), the broad peak at 3425 cm−1 corresponds to the O−H or NeH stretching vibrations, whereas the peaks at 2954 and 1622 cm−1 indicate the existence of C−H and C=O stretching vibration, respectively [35,36]. The absorption and at 1443 cm−1 is assigned to C−N stretching vibration, and that at 1118 cm−1 is attributed to the stretching vibration of C−O [37–39]. Meanwhile, an obvious absorption peak at 270 nm presents in the UV–Vis spectrum of CQDs (Fig. 3(b)), it can be assigned to the n–π* transition of the C=O band and the π–π* transition of the conjugated C=C band [40]. The FTIR, XPS, and UV–Vis results demonstrate that the synthesized CQDs have different kinds of surface chemical bonds such as C=O, O−H, N−H, C−N, and surface functional groups such as −COOH, −OH, −NH2, which enhance their hydrophilicity and stability in aqueous systems. The optical properties of CQDs were also investigated (Fig. 3(b)). And with excitation at 360 nm, CQDs display an emission maximum at 450 nm, which is close to the absorption band in the lower energy region. Besides, the fluorescent spectra of CQDs are excitation-dependent with the increase of excitation wavelength from 310 to 400 nm (Fig. S5), and it may be attributed to the complexity of surface excited states of the CQDs [41]. The QY of CQDs was measured to be 9.01% (Table S1) using quinine sulfate (dissolved in 0.1 M H2SO4) as reference (QY 54% at 360 nm). As the chemical stability and photostability of CQDs play important roles in their applications. Thus, the fluorescence intensity of CQDs depending on several concentrations of NaCl is investigated to evaluate the ionic strength tolerance of the fluorescent probe. And there is nearly no FL signal variation even the concentration of NaCl reach up to 1.0 M (Fig. 4(a)), indicating the CQDs possess good resistance to salt. Subsequently, the influence of pH on the fluorescence of CQDs is also investigated, and the normalized intensity of CQDs mainly keeps stable when the pH value changes from 1 to 8, but it drops remarkably as the solution becomes more alkaline (Fig. 4(b)), suggesting that CQDs can be used at normal physiological range of pH. Besides, to further verify the photostability of CQDs, and the FL intensities of CQDs irradiated with a Xe lamp for different time are also investigated (Fig. 4(c)), and the normalized fluorescence intensity decreases slightly with duration of irradiation. Meanwhile, the fluorescence intensity of CQDs is still above
The HeLa cells (5 × 104 cells/150 μL) were first cultured in an incubator containing Dulbecco's modified Eagle's medium (DMEM) that is supplemented with 10% fetal bovine serum (FBS), penicillin (100 units per mL), and streptomycin (100 units per mL) for 24 h (37 °C and 5% CO2) in a 96-well plate. Then, cells were treated with 100 μL of DMEM containing different concentrations (0, 1, 5, 10, 20, 40, 60, 80 and 100 μg mL−1) of CQDs and incubated for another 24 h. Afterward, 10 μL methylthiazolyldiphenyl-tetrazolium bromide (MTT) solution (5 mg mL−1) was added to each cell wall, and then incubated again for 4 h. After adding100 μL of DMSO into each well to dissolve MTT for three times, the resulting mixture was shaken for 15 min at room temperature in dark. The optical density (OD) of the mixture was recorded at 490 nm using a Thermo Scientific Multiskan spectrum microplate spectrophotometer. The cell viability was calculated according to the following Eq (2) [27].
Cell viability (%) = ODtreated / ODcontrol × 100%
(2)
where ODcontrol was acquired in the absence of CQDs and ODtreated was acquired in the presence of CQDs. The potential of CQDs in biolabeling was preliminarily evaluated by their cellular imaging in Hela cells. The cells (5 × 104 cells) were cultured in a DMEM-supplemented solution containing 10% fetal bovine serum and 1% penicillin/streptomycinin each hole. DMEM medium was used to prepare the CQDs solution with a concentration of 50 μg mL−1 and ultrasonicated for 10 min. Then an aliquot (typically 0.1 mL) of the suspension was added to the well of a chamber slide and incubated at 37 °C for 24 h under a humidified atmosphere containing 5% CO2. The excess CQDs were removed by washing three times with PBS (0.01 M, pH 7.0) buffer. Eventually, the samples were fixed on the slide for analysis by a laser scanning confocal microscope. 3. Results and discussion 3.1. Characterization of CQDs To study the morphology, structure, elements, and surface groups of the prepared CQDs, a series of characterizations including high resolution TEM, XPS, UV–vis, and FT-IR were performed. As the TEM image shown in Fig. 1(a), well monodispersed CQDs have a uniformly spherical shape with the size smaller than 5 nm, and display a narrow size distribution with an average diameter of 2.8 nm (Fig. 1(b)). Besides, the high resolution TEM (HRTEM) image (inserted in Fig. 1(a)) exhibits a clear fringe distance of 0.24 nm, similar to the (1120) lattice fringes of graphene, which indicates the crystalline nature of CQDs [28]. The results of XPS analysis are shown in Fig. 2. Three peaks around 286.0 eV, 532.0 eV, 401.0 eV in Fig. 2(a) can be ascribed to carbon,
Fig. 1. (a) TEM image of CQDs and HRTEM image of CQDs (inset view); (b) The size distribution of the CQDs. 3
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Fig. 2. (a) Full XPS spectrum of CQDs; (b) C 1s XPS spectrum; (c) O 1s XPS spectrum; (d) N1s XPS spectrum of CQDs.
Fig. 3. (a) FT-IR spectrum of CQDs; (b) UV–vis absorption of CQDs (blue) and photoluminescence excitation (red) and emission (black) spectra of CQDs, λex = 340 nm and λem = 420 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
90% after being treated with natural lighting lasting for 30 days in room temperature (Fig. 4(d)), further confirming the remarkable photostability of CQDs. All these studies indicate that the as-prepared CQDs would be appropriate for practical application [42].
spectra of collisional quenching keeps constant before and after quenching. And, as shown in Fig. 5(b), the UV–vis spectrum of CQDsAg+ solution after fluorescence quenching is different from the summed UV–vis absorption spectrum of CQDs and Ag+, continuously elucidating the binding interaction between CQDs and Ag+. Therefore, we can interpret that the fluorescence behavior of the CQDs-Ag+ system is a kind of the static fluorescence quenching. The fluorescence quenching may be attributed to the nonradiative electron-transfer from the excited states to the ｄ orbital of Ag+. Meanwhile, the Ag+-induced conversion of the functional group (−CONH−) from spirolactam structure to an opened-ring amide may also make an important contribution to the fluorescence quenching.
3.2. Fluorescence quenching mechanism There are numerous molecular interactions can result in fluorescence quenching, such as excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and collisional quenching [43]. And those quenching mechanism can be classified into two major categories: dynamic and static quenching, which can be ascertained via measuring of fluorescence lifetime and its variation [44]. As the fluorescence lifetime will be shortened during the process of dynamic quenching, while be invariant in static one [45]. As indicated in Fig. 5(a), the average fluorescence lifetime of CQDs remains unchanged even after the addition of 0.1 or 1 mM Ag+. Besides, UV–vis absorption spectrum of the fluorophore is also investigated to further confirm the fluorescence quenching mechanism, since the absorption
3.3. Optimization of detection conditions Influence of important experimental parameters including pH, reaction time, temperature and concentration on the CQDs based fluorescent sensor is firstly evaluated for better sensing performances. As illustrated in Fig.S1, the fluorescence quenching is pH-dependent, 4
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Fig. 4. Normalized FL intensity of CQDs (a) in presence of various concentrations of NaCl; (b) at different pH; (c) irradiated with a Xe lamp for 60 min and (d) stored at room temperature and under natural lighting for a month, respectively. (The excitation wavelength is 360 nm, the emission wavelength is 450 nm).
which shows the best quenching efficiency at pH 7.0 and low efficiencies at acidic conditions. It may attribute to the hydrolysis and precipitation of Ag+ in weakly acidic solution. Then the kinetic behavior of the detection system is studied to optimize the detection time (Fig. S2). It is evident that the quenching equilibrium is obtained in 15 min and then it remains stable with further increase of incubation time. Besides, the PL quenching efficiency reaches a maximum at 30 °C, then decreases with increasing temperature (Fig. S3). Because the complex at excited state becomes instable at higher temperature, this changing trend also validates that the fluorescence quenching mechanism of CQDs-Ag+ system is a kind of the static fluorescence quenching. Simultaneously, the result reveals that the temperature has a certain influence on the diffusion, activation of the molecule and energy transformation in the interior of molecule as well as a state of
equilibrium of solution. For the optimization of CQDs concentration, as shown in Fig. S4, the FL spectra of CQDs in the existence of Ag+ display a basically linear growth trend in the concentration of CQDs ranging from 20 to 80 μg mL−1. Then the fluorescence increases slowly in the range from 80 to 300 μg mL−1 as a result of the self-quenching effect. The highest fluorescence quenching efficiency is obtained when the concentration of Ag+reaches300 μg mL−1. Thus, the optimized pH 7.0, reaction time 15 min, temperature 30 °C, and concentration of CQDs 300 μg mL−1 are adopted in the following experiments. 3.4. Detection of Ag+ In order to demonstrate the analytical performance of CQDs based fluorescence sensor, the relationship between the concentration of Ag+
Fig. 5. (a) Fluorescence decay traces of CQDs and with addition of different concentration of Ag+ solution; (b) UV–vis absorption spectra of CQDs (blue), Ag+ (green), CQDs-Ag+ system (black), and summed UV–vis absorption spectrum between CQDs and Ag+ (red), respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 5
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Fig. 6. (a) FL spectra of the xCQDs response to Ag+ ions with different concentrations (from top to bottom: 0, 1, 2, 4, 5, 10, 40, 100, 300, 1.0 × 103, 3.0 × 103, 5.0 × 103, 1.0 × 104, 5.0 × 104, 7.0 × 104, 1.0 × 105, 5.0 × 105, 7.0 × 105, 1.0 × 106, 5.0 × 106, 1.0 × 107, 5.0 × 107 and 1.0 × 108 nM); [CQDs] = 300.0 μg mL−1; pH 7.0 of H2O; (b) and (c) Linear relationship between the concentration of Ag+ and the degree of fluorescent quenching (F0-F), F and F0 were the fluorescence intensities of CQDs at 450 nm in the presence or absence of Ag+, respectively; (d) Selectivity for various metal ions (1 mM). Table 1 Comparison of detection performance of different fluorescent probes for Ag+ detection. Detection probes
Detection mechanism
Detection limit
Linear range
Reference
silver nanoclusters and quantum dots Two fluorescent probes based on pyrrolo[2,1-a] isoquinoline N-doped C-dots Graphene quantum dots S-GQDs CQDs
Colorimetric fluorescence quenching PET
32 nM 0.6 μM, 0.8 μM
– 0–30 μM
[49] [50]
Fluorescence Fluorescence Fluorescence Fluorescence
enhancement quenching quenching quenching
1 μM 0.3 mM 30 nM 1.4 nM
1–100 μM – 0.1–130 nM 0–10 nM,10–3000 nM
[46] [47] [48] This work
Table 2 Determination of Ag+ in real water samples. Samples
No.
Added (nM)
Found (nM)
Recovery (%)
RSD (%) (n = 3)
Xiangjiang river of Changsha
1 2 3 4 1 2 3 4 1 2 3 4
0 50 100 150 0 50 100 150 0 50 100 150
1520 1572 1623 1669 1000 1049 1105 1152 – 56 99 154
– 104 103 99.3 – 98.0 105 101 – 112 99.0 103
0.51 0.65 0.58 0.59 0.52 0.46 0.64 0.76 0.44 0.69 0.56 0.72
Yudai river ofChangsha
Tap water oflaboratory
Fig. 7. Effects of CQDs at different concentrations on the viability of Hela cells for 24 h. 6
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Fig. 8. HeLa cells treated with 50 μg mL−1 CQDs solution. (a) Bright field image of HeLa cells; (b) Fluorescence image of HeLa cells (blue channel); (c) Overlay of fluorescence and bright-field images of HeLa cells. Scale bar: 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
and the degree of fluorescent quenching (F0-F) was studied. As shown in Fig. 6(a), the fluorescence intensity of CQDs at around 450 nm decreases gradually with increase of Ag+ concentration, indicating that addition of Ag+ can effectively quench the fluorescence of CQDs. And the fluorescence intensities of CQDs are weakened gradually as the concentration of Ag+ increases from 0.5 to 1.0 × 108 nM. As shown in Fig. 6(b) and (c), good linear correlations (y = 1.650x + 7.618, y = 0.03901x + 25.97) over the range from 0.5 to 10 nM and 10–3000 nM with correlation coefficient squares (R2) 0.9952 and 0.9802 are obtained. The limit of detection (LOD) is calculated to be 1.4 nM (S/N = 3), which is much lower than that of previously reports [46–48] (Table 1). More importantly, compared with reported semiconductor QDs [49] and conventional dyes CQDs [50], the purple perilla-derived CQDs exhibit several advantages including bright fluorescence, high photostability, and good water solubility. To sum up, purple perilla-based CQDs were innovatively developed as an effective “signal-off” fluorescent probe for highly sensitive detection of silver ion with wide ranges and especially the lowest detection limit in this work. Then, the selectivity of proposed method is evaluated by challenging the detection with other twenty-five ions. As depicted in Fig. 6(d), the normalized fluorescence intensity of the CQDs is remarkably quenched only in the presence of Ag+, whereas no significant fluorescence quenching is noticed with other environmentally competing chemical species. Hence, Ag+ has the highest selectivity towards the fluorescence quenching of CQDs, implying that CQDs could be used to selectively detect Ag+ in aqueous solution. To exploit the practical application of CQDs as fluorescence probes in detection of silver ion, standard addition experiments are performed with water samples to validate this method. As shown in Table 2, the relative standard deviation (RSD) is lower than 0.89 and the recoveries are between 98% and 108% for the samples from Xiangjiang River, Yudai River and tap water of laboratory. These results exemplify the potential applicability of the CQDs-based fluorescent probe in the detection of Ag+ in real water samples.
low. Then the cytotoxicity of the fluorescent probe is further verified via endocytosis of CQDs in living cells. Fig. 8(a) shows confocal image of HeLa cells under bright-field illumination. It is clearly observed obvious blue fluorescence in HeLa cells under 346 nm laser excitation (Fig. 8(b)). As shown in Fig. 8(c), the merged image indicates the ability of CQDs to penetrate through the cell membrane without any additional surface passivating treatment. Significantly, the experiment demonstrates that CQDs can easily permeate through the cell membrane causing no harm to the cells. 4. Conclusions In this work, CQDs with fine optical stabilities are synthesized deriving from purple perilla through a facile one-step hydrothermal treatment for the first time. The obtained CQDs are thoroughly characterized to confirm the surface functional groups of CQDs. And based on the biomass-derived CQDs, a fluorescent sensor for sensitive and selective detection of Ag+ is favorably developed as the fluorescence intensity of CQDs can be effectively quenched by Ag+. Compared with other fluorescence probes such as conventional dyes, semiconductor QDs and CQDs-based composite material, the purple perilla-based CQDs developed in this work not only have low cytotoxicity, high photostability, good water solubility and biocompatibility, but also exhibit excellent fluorescence response for Ag+ with wide ranges and especially at a low detection limit. Meanwhile, the fluorescence quenching is also revealed following a static mechanism. We believe that the remarkable biocompatibility, good photo-stability of CQDs promise its potential application in imaging of living cells and as an effective probe for exploring inexpensive, highly sensitive and selective sensors in environmental and biological scenarios. Conflict of interest The authors have declared no conflict of interest.
3.5. Cytotoxicity studies and bioimaging
Acknowledgments
The cytotoxicity of CQDs is evaluated via MTT assay, where HeLa cells are incubated with different concentrations of CQDs for 24 h. As the results shown in Fig. 7, high cell viability over 95% is consistently observed for the tested CQDs solutions even the concentration reaches up to 100.0 μg mL−1, indicating the cytotoxicity of CQDs is relatively
We gratefully acknowledge the financial support from National Natural Science Foundation of China(No. 21576296 and No. 21878339) and Key Laboratory of Hunan Province for Water Environment and Agriculture Product Safety (No. 2018TP1003).
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.talanta.2019.03.095. The recovery rates are calculated according the equation as follows:
recovery = (found
foundblack )/ added × 100%.
where the subscript “black” (foundblack) refers to the concentration of Ag+ detected in non-spiked real samples. So, when “added” is zero, “found” is equal to foundblack concerning Xiangjiang river of Changsha and Yudai river of Changsha, which are 1520 and 1000 nM respectively. But for tap water of laboratory, when “added” is zero, the concentration of Ag+ could not been detected, that is, “foundblack” is zero. 7
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References
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Facile green and one-pot synthesis of purple perilla derived carbon quantum dot as a fluorescent sensor for silver ion
T
Xinyi Zhaoa, Sen Liaoa, Lumin Wanga, Qi Liua,∗∗, Xiaoqing Chena,b,∗ a b
College of Chemistry and Chemical Engineering, Central South University, Changsha, 410083, Hunan, China Key Laboratory of Hunan Province for Water Environment and Agriculture Product Safety, Central South University, Changsha, 410083, Hunan, China
ABSTRACT
In this work, biomass-derived carbon quantum dots (CQDs) with excellent water solubility, strong fluorescence and favorable biocompatibility were synthesized via one-step hydrothermal treatment of purple perilla for the first time. The functional group composition, morphology, and pH stability of the synthesized CQDs were systematically investigated. And based on fluorescence quenching of CQDs, the as-prepared CQDs were innovatively developed as an effective “signal-off” fluorescent probe for selective and sensitive detection of silver ion (Ag+) with two linear ranges of 0–10 and 10–3000 nM, and a detection limit 1.4 nM. The specificity and selectivity of this fluorescent probe were also verified through challenging the detection by using similarmetallic cations or in real water samples. In addition, the asprepared CQDs exhibit a low cytotoxicity and a good biocompatibility, revealing its potential bioimaging applications in living cells.
1. Introduction Resulting from its advantages including high quantum yield, well water solubility, excellent chemical stability, good biocompatibility and ease of modification, carbon quantum dots (CQDs) have recently emerged as a new nanocarbon materials used for bioimaging, biosensing, drug delivery, efficient visible light-active photocatalysts and so forth [1–7]. Generally, the synthetic method to produce CQDs can be divided into two broad categories, namely bottom-up and top-down. And production of CQDs can be realized through synthetic approaches such as arc-discharge, laser ablation, electrochemical, hydrothermal, ultrasonic and microwave treatment [8–12]. However, the majority of above approaches have some limitations, such as limited spectral efficiency, low product yield, lack of size control, and the use of toxic chemicals or high temperature for experiments. Hydrothermal carbonization has arisen as a powerful and sustainable technology for the synthesis of CQDs in aqueous media since it is green, facile, convenient and efficient. After first biomass-derived CQDs obtained by hydrothermal treatment of grass in 2012 [13], more attentions have been paid to producing CQDs from natural plants, such as cabbage, sweet red pepper, aloe and various vegetables [14]. Especially, biomass-derived CQDs can be easily fabricated by using the variety of natural carbon sources. It also can effectively avoid the use of costly/toxic chemicals and complicated post-treatment processes. In addition, these CQDs derived from biomass have numerbers of advantages including cheap raw materials, easy contronl, massproduction and high yield. Moreover,
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bioprotein [14–16] and wastes (such as waste pololefins [17], fish scales [18] and even human urine [19]) also can be used as carbon sources for preparing CQDs. In summary, there are multiple inherent advantages in reusing the waste materials and natural bioresources, such as abundance of carbon sources from low-value precursor materials, varieties of heteroatom doping (such as N, S, P), saving of chemical precursors, addressing of environmental issues caused by waste disposal etc [17,20]. Perilla frutescens (L.) Britt. referred to as purple perilla (also called Zisu in China), belonging to the family Labiatae, is distributed worldwide, especially in China, Japan, Korea, and other regions in Asia [21]. In China, the plant of purple perilla is not only used as a traditionally medicinal herb for various diseases, but also as a common flavor for fish and crab cooking with the purposes of detoxification. Till now, various compounds including flavonoids, volatile oils, fatty acids, triterpenes, phenolic compounds have been isolated and identified from this plant, which can serve as an excellent source of carbon and nitrogen to produce CQDs. And compared with organic molecules as precursor to synthesize CQDs, the natural source of carbon and nitrogen from purple perilla possess great characteristics including convenient sources, low price, good water solubility, innocuity and no pollution to environment. Silver ion (Ag+) is one of several essential trace element species in the human body, and toxicity acts as a stimulant for the production of red blood cells. Nevertheless, high level of Ag+ in organisms might cause serious consequences. And the potential toxicity is continuously escalated as the Ag+ being accumulated by aquatic organisms to poison
Corresponding author. College of Chemistry and Chemical Engineering, Central South University, Changsha, 410083, Hunan, China. Corresponding author. College of Chemistry and Chemical Engineering, Central South University, Changsha, 410083, Hunan, China. E-mail addresses: [email protected] (Q. Liu), [email protected] (X. Chen).
∗∗
https://doi.org/10.1016/j.talanta.2019.03.095 Received 30 September 2018; Received in revised form 22 March 2019; Accepted 27 March 2019 Available online 30 March 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
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Scheme 1. Illustration of the synthetic procedure of the CQDs and the CQDs-based fluorescence biosensing platform for the detection of Ag+.
aquatic animals, plants, and even human body in previously reports [22]. Due to its extreme toxicity, the maximum permissive level of Ag+ in drinking water is set at 0.05 ppm by the World Health Organization [23]. Consequently, monitoring the levels of Ag+ in the environment is deemed important for environmental protection and health reasons. Till now, methods such as atomic absorption spectroscopy [24], electrochemistry [25], fluorescence, and inductively coupled plasma mass spectroscopy have been used to detect metal ions. In which, the fluorescence analysis method is currently considered to be a highly effective way for Ag+ detection. Therefore, it is of great significance to design and prepare a highly sensitive and selective fluorescent probe for Ag+ detection. Compared with other fluorescent materials (conventional dyes, polymers, and semiconductor quantum dots), CQDs could offer the advantageous features of bright fluorescence, high photostability, low toxicity, and resistance to metabolic degradation in bio-applications [26]. In the present study, purple perilla was selected as the source for the facile one-pot synthesis of biomass-derived CQDs for the first time. And, as shown in Scheme 1, the resulting blue fluorescent CQDs was then served as the probe fordetection of silver ion because the luminescence can be effectively quenched by the target (Ag+). The specificity and selectivity of the CQDs probe were also evaluated via challenging the detection by using other metal cations or in complicated lake water. Besides, the related mechanisms of the CQDs formation and the Ag+-induced fluorescence quenching were also illustrated.
2600 UV–Vis spectrophotometer (Shimadzu, Japan) was introduced for UV–vis absorption measurements. The Zeta potentials were measured on a Zetasizer Nano ZS (Malvern Instruments Ltd., United Kingdom) equipped with a noninvasive back scattering (NIBS) device and folded capillary sample cell (for Zeta potentials). 2.3. Synthesis of CQDs CQDs were synthesized via a one-step hydrothermal treatment of purple perilla in Teflon lined autoclave. Initially, dried purple perilla was purchased from a local Herbalists and smashed by a domestic pulverizer. And the moderate power of purple perilla was added to 30 mL of distilled water in a Teflon lined hydrothermal reactor. Then the solution was transferred into a 50 mL poly(tetrafluoroethylene)-lined auto-clave after the ultrasonic treatment for a period of time, and heated at 260 °C for 5 h. After being cooled down to room temperature, the light brown suspension was collected through a 0.22 μm micro-pore film filter to eliminate the unreacted raw materials and the large dots. Finally, the obtained CQDs were dialyzed against ultrapure water through a dialysis membrane (MWAG of 100 Da) for 2 days to remove impurities while ultrapure water was changed with an interval of 8 h. Then the dispersion was stored at 4 °C. 2.4. Detection of Ag+
2. Experimental
CQDs was adjusted to a concentration of 300 μg mL−1 in 0.1 mM aqueous solutions containing twenty-five different ions (including Ag+, Co2+, Mg2+, Hg2+, Ni2+, Sr2+, Ca2+, Pb2+, Li+, Zn2+, Cu2+, Cd2+, Fe3+, Fe2+, Cr3+, Al3+, Mn2+, Na+, K+, SO42−, Cl−, S2−, NO3−, CO32−, SO32−), and then subjected it to fluorescence measurements after 15 min. The quenching effect of Ag+ on fluorescence intensity of CQDs was conducted as follows. Typically, 1 mL of CQDs aqueous solution (300 μg mL−1 in deionized water, pH 7.0) was placed in each of 4.0 mL EP tubes, and then the final aqueous systems were acquired by the addition of a calculated amount of Ag+ stock solution, respectively. The final concentrations of Ag+ presented were from 0.5 to 1.0 × 108 nM. The fluorescence emission intensity at 450 nm of the blank sample excited at 360 nm were measured and marked as F0. After equilibration for 15 min, the fluorescent intensity (F) of each sample was measured.
2.1. Materials and reagents Dried purple perilla was bought from local Herbalists (Changsha, China). Quinine sulfate was purchased from Aladdin (Shanghai, China). Na2SO4, Na2SO3, CoSO4, AgNO3, Cu(NO3)2, Pb(NO3)2, LiCl, Zn(NO3)2, Cd(NO3)2, NiSO4, MgSO4, CaCl2, SrCl2, HgCl2, FeCl3.6H2O, FeSO4, MnCl2, CrCl3, AlCl3, KCl, NaCl, Na2S, NaNO3 and Na2CO3 were supplied by Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). All reagents were analytical reagent grade. All aqueous solutions were diluted by ultrapure water with a resistivity≥ 18.2 MΩ cm−1 form a Molecular water purification system. 2.2. Instruments
2.5. Analysis of practical samples
Transmission electron microscope (TEM) images displaying the morphological evaluation and sizes of CQDs were analyzed by a HT7700 transmission electron microscope (TEM, Hitachi) under an accelerating voltage of 200 kV. The surface functional properties of CQDs were investigated by PHI Quantera Ⅱ X-ray photoelectron spectroscopy (XPS, Thermo Fisher Scientific 250Xi). Infrared spectra were obtained using IR Prestige-21 Fourier transform infrared (FTIR) (Shimadzu, Japan). The fluorescence and time-resolved fluorescence decay tests were conducted using a LS-55 spectrofluorometer (PerkinElmer, America) and a Fluo Time 100 compact fluorescence lifetime spectrophotometer (PicoQuant, Germany), respectively. A UV-
To evaluate the viability and practicality of the developed CQDs sensor for Ag+ detection in practical water samples, the performance of the current method was examined by lake water samples which obtained from Xiangjiang River of Changsha (China) and tap water samples from our lab. The obtained real water samples were filtered through 0.22 μm micro-pore film filter. The river water was added standard Ag+ solutions with different concentrations levels, which was analyzed by the proposed method. The tap water samples were spiked with the different concentrations of Ag+ solution directly without any pretreatment. 2
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2.6. Cytotoxicity assay and cellular imaging of CQDs
oxygen and nitrogen, respectively [29]. And there are four main peaks in the high-resolution spectrum of C 1s in Fig. 2(b), indicating the presence of four types of carbon bonds, corresponding to sp2 C (C−C or C=C) in graphene at 284.5 eV, sp3 C from C−N and C−O at 285.5 eV, C=N at 287.1 eV, and C=O at 288.7 eV [30,31]. Besides, as displayed in Fig. 2(c), there are two peaks 531.9 and 532.9 eV present in the O 1s spectrum, which can be assigned as the C=O and C–OH groups, respectively [32,33]. Additionally, deconvolution of N 1s spectra in Fig. 2(d) reveals two relative nitrogen species of N−H (400.1 eV) and C−N (401.4 eV) [34]. As for the FT-IR spectrum of CQDs (Fig. 3(a)), the broad peak at 3425 cm−1 corresponds to the O−H or NeH stretching vibrations, whereas the peaks at 2954 and 1622 cm−1 indicate the existence of C−H and C=O stretching vibration, respectively [35,36]. The absorption and at 1443 cm−1 is assigned to C−N stretching vibration, and that at 1118 cm−1 is attributed to the stretching vibration of C−O [37–39]. Meanwhile, an obvious absorption peak at 270 nm presents in the UV–Vis spectrum of CQDs (Fig. 3(b)), it can be assigned to the n–π* transition of the C=O band and the π–π* transition of the conjugated C=C band [40]. The FTIR, XPS, and UV–Vis results demonstrate that the synthesized CQDs have different kinds of surface chemical bonds such as C=O, O−H, N−H, C−N, and surface functional groups such as −COOH, −OH, −NH2, which enhance their hydrophilicity and stability in aqueous systems. The optical properties of CQDs were also investigated (Fig. 3(b)). And with excitation at 360 nm, CQDs display an emission maximum at 450 nm, which is close to the absorption band in the lower energy region. Besides, the fluorescent spectra of CQDs are excitation-dependent with the increase of excitation wavelength from 310 to 400 nm (Fig. S5), and it may be attributed to the complexity of surface excited states of the CQDs [41]. The QY of CQDs was measured to be 9.01% (Table S1) using quinine sulfate (dissolved in 0.1 M H2SO4) as reference (QY 54% at 360 nm). As the chemical stability and photostability of CQDs play important roles in their applications. Thus, the fluorescence intensity of CQDs depending on several concentrations of NaCl is investigated to evaluate the ionic strength tolerance of the fluorescent probe. And there is nearly no FL signal variation even the concentration of NaCl reach up to 1.0 M (Fig. 4(a)), indicating the CQDs possess good resistance to salt. Subsequently, the influence of pH on the fluorescence of CQDs is also investigated, and the normalized intensity of CQDs mainly keeps stable when the pH value changes from 1 to 8, but it drops remarkably as the solution becomes more alkaline (Fig. 4(b)), suggesting that CQDs can be used at normal physiological range of pH. Besides, to further verify the photostability of CQDs, and the FL intensities of CQDs irradiated with a Xe lamp for different time are also investigated (Fig. 4(c)), and the normalized fluorescence intensity decreases slightly with duration of irradiation. Meanwhile, the fluorescence intensity of CQDs is still above
The HeLa cells (5 × 104 cells/150 μL) were first cultured in an incubator containing Dulbecco's modified Eagle's medium (DMEM) that is supplemented with 10% fetal bovine serum (FBS), penicillin (100 units per mL), and streptomycin (100 units per mL) for 24 h (37 °C and 5% CO2) in a 96-well plate. Then, cells were treated with 100 μL of DMEM containing different concentrations (0, 1, 5, 10, 20, 40, 60, 80 and 100 μg mL−1) of CQDs and incubated for another 24 h. Afterward, 10 μL methylthiazolyldiphenyl-tetrazolium bromide (MTT) solution (5 mg mL−1) was added to each cell wall, and then incubated again for 4 h. After adding100 μL of DMSO into each well to dissolve MTT for three times, the resulting mixture was shaken for 15 min at room temperature in dark. The optical density (OD) of the mixture was recorded at 490 nm using a Thermo Scientific Multiskan spectrum microplate spectrophotometer. The cell viability was calculated according to the following Eq (2) [27].
Cell viability (%) = ODtreated / ODcontrol × 100%
(2)
where ODcontrol was acquired in the absence of CQDs and ODtreated was acquired in the presence of CQDs. The potential of CQDs in biolabeling was preliminarily evaluated by their cellular imaging in Hela cells. The cells (5 × 104 cells) were cultured in a DMEM-supplemented solution containing 10% fetal bovine serum and 1% penicillin/streptomycinin each hole. DMEM medium was used to prepare the CQDs solution with a concentration of 50 μg mL−1 and ultrasonicated for 10 min. Then an aliquot (typically 0.1 mL) of the suspension was added to the well of a chamber slide and incubated at 37 °C for 24 h under a humidified atmosphere containing 5% CO2. The excess CQDs were removed by washing three times with PBS (0.01 M, pH 7.0) buffer. Eventually, the samples were fixed on the slide for analysis by a laser scanning confocal microscope. 3. Results and discussion 3.1. Characterization of CQDs To study the morphology, structure, elements, and surface groups of the prepared CQDs, a series of characterizations including high resolution TEM, XPS, UV–vis, and FT-IR were performed. As the TEM image shown in Fig. 1(a), well monodispersed CQDs have a uniformly spherical shape with the size smaller than 5 nm, and display a narrow size distribution with an average diameter of 2.8 nm (Fig. 1(b)). Besides, the high resolution TEM (HRTEM) image (inserted in Fig. 1(a)) exhibits a clear fringe distance of 0.24 nm, similar to the (1120) lattice fringes of graphene, which indicates the crystalline nature of CQDs [28]. The results of XPS analysis are shown in Fig. 2. Three peaks around 286.0 eV, 532.0 eV, 401.0 eV in Fig. 2(a) can be ascribed to carbon,
Fig. 1. (a) TEM image of CQDs and HRTEM image of CQDs (inset view); (b) The size distribution of the CQDs. 3
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Fig. 2. (a) Full XPS spectrum of CQDs; (b) C 1s XPS spectrum; (c) O 1s XPS spectrum; (d) N1s XPS spectrum of CQDs.
Fig. 3. (a) FT-IR spectrum of CQDs; (b) UV–vis absorption of CQDs (blue) and photoluminescence excitation (red) and emission (black) spectra of CQDs, λex = 340 nm and λem = 420 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
90% after being treated with natural lighting lasting for 30 days in room temperature (Fig. 4(d)), further confirming the remarkable photostability of CQDs. All these studies indicate that the as-prepared CQDs would be appropriate for practical application [42].
spectra of collisional quenching keeps constant before and after quenching. And, as shown in Fig. 5(b), the UV–vis spectrum of CQDsAg+ solution after fluorescence quenching is different from the summed UV–vis absorption spectrum of CQDs and Ag+, continuously elucidating the binding interaction between CQDs and Ag+. Therefore, we can interpret that the fluorescence behavior of the CQDs-Ag+ system is a kind of the static fluorescence quenching. The fluorescence quenching may be attributed to the nonradiative electron-transfer from the excited states to the ｄ orbital of Ag+. Meanwhile, the Ag+-induced conversion of the functional group (−CONH−) from spirolactam structure to an opened-ring amide may also make an important contribution to the fluorescence quenching.
3.2. Fluorescence quenching mechanism There are numerous molecular interactions can result in fluorescence quenching, such as excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and collisional quenching [43]. And those quenching mechanism can be classified into two major categories: dynamic and static quenching, which can be ascertained via measuring of fluorescence lifetime and its variation [44]. As the fluorescence lifetime will be shortened during the process of dynamic quenching, while be invariant in static one [45]. As indicated in Fig. 5(a), the average fluorescence lifetime of CQDs remains unchanged even after the addition of 0.1 or 1 mM Ag+. Besides, UV–vis absorption spectrum of the fluorophore is also investigated to further confirm the fluorescence quenching mechanism, since the absorption
3.3. Optimization of detection conditions Influence of important experimental parameters including pH, reaction time, temperature and concentration on the CQDs based fluorescent sensor is firstly evaluated for better sensing performances. As illustrated in Fig.S1, the fluorescence quenching is pH-dependent, 4
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Fig. 4. Normalized FL intensity of CQDs (a) in presence of various concentrations of NaCl; (b) at different pH; (c) irradiated with a Xe lamp for 60 min and (d) stored at room temperature and under natural lighting for a month, respectively. (The excitation wavelength is 360 nm, the emission wavelength is 450 nm).
which shows the best quenching efficiency at pH 7.0 and low efficiencies at acidic conditions. It may attribute to the hydrolysis and precipitation of Ag+ in weakly acidic solution. Then the kinetic behavior of the detection system is studied to optimize the detection time (Fig. S2). It is evident that the quenching equilibrium is obtained in 15 min and then it remains stable with further increase of incubation time. Besides, the PL quenching efficiency reaches a maximum at 30 °C, then decreases with increasing temperature (Fig. S3). Because the complex at excited state becomes instable at higher temperature, this changing trend also validates that the fluorescence quenching mechanism of CQDs-Ag+ system is a kind of the static fluorescence quenching. Simultaneously, the result reveals that the temperature has a certain influence on the diffusion, activation of the molecule and energy transformation in the interior of molecule as well as a state of
equilibrium of solution. For the optimization of CQDs concentration, as shown in Fig. S4, the FL spectra of CQDs in the existence of Ag+ display a basically linear growth trend in the concentration of CQDs ranging from 20 to 80 μg mL−1. Then the fluorescence increases slowly in the range from 80 to 300 μg mL−1 as a result of the self-quenching effect. The highest fluorescence quenching efficiency is obtained when the concentration of Ag+reaches300 μg mL−1. Thus, the optimized pH 7.0, reaction time 15 min, temperature 30 °C, and concentration of CQDs 300 μg mL−1 are adopted in the following experiments. 3.4. Detection of Ag+ In order to demonstrate the analytical performance of CQDs based fluorescence sensor, the relationship between the concentration of Ag+
Fig. 5. (a) Fluorescence decay traces of CQDs and with addition of different concentration of Ag+ solution; (b) UV–vis absorption spectra of CQDs (blue), Ag+ (green), CQDs-Ag+ system (black), and summed UV–vis absorption spectrum between CQDs and Ag+ (red), respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 5
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Fig. 6. (a) FL spectra of the xCQDs response to Ag+ ions with different concentrations (from top to bottom: 0, 1, 2, 4, 5, 10, 40, 100, 300, 1.0 × 103, 3.0 × 103, 5.0 × 103, 1.0 × 104, 5.0 × 104, 7.0 × 104, 1.0 × 105, 5.0 × 105, 7.0 × 105, 1.0 × 106, 5.0 × 106, 1.0 × 107, 5.0 × 107 and 1.0 × 108 nM); [CQDs] = 300.0 μg mL−1; pH 7.0 of H2O; (b) and (c) Linear relationship between the concentration of Ag+ and the degree of fluorescent quenching (F0-F), F and F0 were the fluorescence intensities of CQDs at 450 nm in the presence or absence of Ag+, respectively; (d) Selectivity for various metal ions (1 mM). Table 1 Comparison of detection performance of different fluorescent probes for Ag+ detection. Detection probes
Detection mechanism
Detection limit
Linear range
Reference
silver nanoclusters and quantum dots Two fluorescent probes based on pyrrolo[2,1-a] isoquinoline N-doped C-dots Graphene quantum dots S-GQDs CQDs
Colorimetric fluorescence quenching PET
32 nM 0.6 μM, 0.8 μM
– 0–30 μM
[49] [50]
Fluorescence Fluorescence Fluorescence Fluorescence
enhancement quenching quenching quenching
1 μM 0.3 mM 30 nM 1.4 nM
1–100 μM – 0.1–130 nM 0–10 nM,10–3000 nM
[46] [47] [48] This work
Table 2 Determination of Ag+ in real water samples. Samples
No.
Added (nM)
Found (nM)
Recovery (%)
RSD (%) (n = 3)
Xiangjiang river of Changsha
1 2 3 4 1 2 3 4 1 2 3 4
0 50 100 150 0 50 100 150 0 50 100 150
1520 1572 1623 1669 1000 1049 1105 1152 – 56 99 154
– 104 103 99.3 – 98.0 105 101 – 112 99.0 103
0.51 0.65 0.58 0.59 0.52 0.46 0.64 0.76 0.44 0.69 0.56 0.72
Yudai river ofChangsha
Tap water oflaboratory
Fig. 7. Effects of CQDs at different concentrations on the viability of Hela cells for 24 h. 6
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Fig. 8. HeLa cells treated with 50 μg mL−1 CQDs solution. (a) Bright field image of HeLa cells; (b) Fluorescence image of HeLa cells (blue channel); (c) Overlay of fluorescence and bright-field images of HeLa cells. Scale bar: 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
and the degree of fluorescent quenching (F0-F) was studied. As shown in Fig. 6(a), the fluorescence intensity of CQDs at around 450 nm decreases gradually with increase of Ag+ concentration, indicating that addition of Ag+ can effectively quench the fluorescence of CQDs. And the fluorescence intensities of CQDs are weakened gradually as the concentration of Ag+ increases from 0.5 to 1.0 × 108 nM. As shown in Fig. 6(b) and (c), good linear correlations (y = 1.650x + 7.618, y = 0.03901x + 25.97) over the range from 0.5 to 10 nM and 10–3000 nM with correlation coefficient squares (R2) 0.9952 and 0.9802 are obtained. The limit of detection (LOD) is calculated to be 1.4 nM (S/N = 3), which is much lower than that of previously reports [46–48] (Table 1). More importantly, compared with reported semiconductor QDs [49] and conventional dyes CQDs [50], the purple perilla-derived CQDs exhibit several advantages including bright fluorescence, high photostability, and good water solubility. To sum up, purple perilla-based CQDs were innovatively developed as an effective “signal-off” fluorescent probe for highly sensitive detection of silver ion with wide ranges and especially the lowest detection limit in this work. Then, the selectivity of proposed method is evaluated by challenging the detection with other twenty-five ions. As depicted in Fig. 6(d), the normalized fluorescence intensity of the CQDs is remarkably quenched only in the presence of Ag+, whereas no significant fluorescence quenching is noticed with other environmentally competing chemical species. Hence, Ag+ has the highest selectivity towards the fluorescence quenching of CQDs, implying that CQDs could be used to selectively detect Ag+ in aqueous solution. To exploit the practical application of CQDs as fluorescence probes in detection of silver ion, standard addition experiments are performed with water samples to validate this method. As shown in Table 2, the relative standard deviation (RSD) is lower than 0.89 and the recoveries are between 98% and 108% for the samples from Xiangjiang River, Yudai River and tap water of laboratory. These results exemplify the potential applicability of the CQDs-based fluorescent probe in the detection of Ag+ in real water samples.
low. Then the cytotoxicity of the fluorescent probe is further verified via endocytosis of CQDs in living cells. Fig. 8(a) shows confocal image of HeLa cells under bright-field illumination. It is clearly observed obvious blue fluorescence in HeLa cells under 346 nm laser excitation (Fig. 8(b)). As shown in Fig. 8(c), the merged image indicates the ability of CQDs to penetrate through the cell membrane without any additional surface passivating treatment. Significantly, the experiment demonstrates that CQDs can easily permeate through the cell membrane causing no harm to the cells. 4. Conclusions In this work, CQDs with fine optical stabilities are synthesized deriving from purple perilla through a facile one-step hydrothermal treatment for the first time. The obtained CQDs are thoroughly characterized to confirm the surface functional groups of CQDs. And based on the biomass-derived CQDs, a fluorescent sensor for sensitive and selective detection of Ag+ is favorably developed as the fluorescence intensity of CQDs can be effectively quenched by Ag+. Compared with other fluorescence probes such as conventional dyes, semiconductor QDs and CQDs-based composite material, the purple perilla-based CQDs developed in this work not only have low cytotoxicity, high photostability, good water solubility and biocompatibility, but also exhibit excellent fluorescence response for Ag+ with wide ranges and especially at a low detection limit. Meanwhile, the fluorescence quenching is also revealed following a static mechanism. We believe that the remarkable biocompatibility, good photo-stability of CQDs promise its potential application in imaging of living cells and as an effective probe for exploring inexpensive, highly sensitive and selective sensors in environmental and biological scenarios. Conflict of interest The authors have declared no conflict of interest.
3.5. Cytotoxicity studies and bioimaging
Acknowledgments
The cytotoxicity of CQDs is evaluated via MTT assay, where HeLa cells are incubated with different concentrations of CQDs for 24 h. As the results shown in Fig. 7, high cell viability over 95% is consistently observed for the tested CQDs solutions even the concentration reaches up to 100.0 μg mL−1, indicating the cytotoxicity of CQDs is relatively
We gratefully acknowledge the financial support from National Natural Science Foundation of China(No. 21576296 and No. 21878339) and Key Laboratory of Hunan Province for Water Environment and Agriculture Product Safety (No. 2018TP1003).
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.talanta.2019.03.095. The recovery rates are calculated according the equation as follows:
recovery = (found
foundblack )/ added × 100%.
where the subscript “black” (foundblack) refers to the concentration of Ag+ detected in non-spiked real samples. So, when “added” is zero, “found” is equal to foundblack concerning Xiangjiang river of Changsha and Yudai river of Changsha, which are 1520 and 1000 nM respectively. But for tap water of laboratory, when “added” is zero, the concentration of Ag+ could not been detected, that is, “foundblack” is zero. 7
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