Accepted Manuscript A sensitive fluorescent sensor for selective determination of dichlorvos based on the recovered fluorescence of carbon dots-Cu(II) system Juying Hou, Guangjuan Dong, Zhengbin Tian, Jiutian Lu, Qianqian Wang, Shiyun Ai, Minglin Wang PII: DOI: Reference:
S0308-8146(15)30268-5 http://dx.doi.org/10.1016/j.foodchem.2015.11.134 FOCH 18475
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
28 July 2015 2 November 2015 28 November 2015
Please cite this article as: Hou, J., Dong, G., Tian, Z., Lu, J., Wang, Q., Ai, S., Wang, M., A sensitive fluorescent sensor for selective determination of dichlorvos based on the recovered fluorescence of carbon dots-Cu(II) system, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/j.foodchem.2015.11.134
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A sensitive fluorescent sensor for selective determination of dichlorvos based on the recovered fluorescence of carbon dots-Cu(II) system Juying Hou a,b, Guangjuan Donga, Zhengbin Tiana, Jiutian Lua, Qianqian Wanga, Shiyun Ai*,a, Minglin Wang*,b a
College of Chemistry and Material Science, Shandong Agricultural University,
Taian, Shandong 271018, P.R. China b
College of Food Science and Engineering, Shandong Agricultural University, Taian,
Shandong 271018, P.R. China
*
Corresponding authors:
Tel: +86 538 8247660 Fax: +86 538 8242251 E-mail address: [email protected] (S.Y. Ai) [email protected] (M.L. Wang) 1
Abstract In this paper, a simple and sensitive fluorescent sensor for dichlorvos was first constructed based on carbon dots-Cu(II) system. These carbon dots were obtained by simple hydrothermal reaction of feather. The fluorescence of these carbon dots can be selectively quenched by Cu2+ ion. When acetylcholinesterase and acetylthiocholine were introduced into the system, thiocholine came into being, which can react with Cu2+ ion and restore the fluorescence of the system. The reaction mechanism between Cu2+ ion and thiocholine was confirmed by X-ray photoelectron spectroscopy. As one kind of acetylcholinesterase inhibitor, organophosphorus pesticides can be detected based on this sensing system. As an example of organophosphorus pesticides, dichlorvos was detected with a linear range of 6.0 × 10-9 - 6.0 × 10 -8 M. This sensing system has been successfully used for the analysis of cabbage and fruit juice samples. Keywords: Carbon dots; Fluorescent; Sensor; Organophosphorus compounds; Acetylcholinesterase; Enzyme inhibition 1. Introduction Organophosphorus compounds (OPs) have been extensively used as pesticides in modern agriculture. As estimated, millions of tons of organophosphate pesticides are used in agriculture each year, which can raise serious human health and environmental concerns. More than two million people die from OPs poisoning every year. Therefore, there is an urgent demand for the determination of OPs. Conventional analytical techniques, such as liquid chromatography, gas chromatography and tandem mass spectrometer (Amini, Shariatgorji, Crescenzi, & Thorsén, 2009; Lambropoulou & Albanis, 2007; Zhou, Xiao, & Li, 2012) need expensive 2
equipments, skilled personnel and time-consuming treatment of samples. Owing to high sensitivity, low-cost, and operational rapidity and simplicity, fluorescent detection have attracted tremendous attention (Azab, Duerkop, Anwar, Hussein, Rizk, & Amin, 2013; Gao, Tang, & Su, 2012; Long, Li, Zhang, & Yao, 2015; Meng, Wei, Ren, Ren, & Tang, 2013; K. Zhang, Yu, Liu, Sun, Yu, Liu, et al., 2014). However, fluorescence materials using in these methods suffered from some disadvantages, such as tedious preparation procedures, poisonous synthetic conditions, and toxicity of material etc. Recently, fluorescent carbon dots (CDs) have attracted tremendous research interests (K. Jiang, Sun, Zhang, Lu, Wu, Cai, et al., 2015; J. Wang, Zhang, Huang, Liu, Leung, & Wáng, 2015; Xu, Pu, Zhao, Dong, Gao, Chen, et al., 2015; L. Zhang, Han, Zhu, Zhai, & Dong, 2015), owing to their appealing advantages, such as good water-solubility, high chemical inertness, good photostability, low cytotoxicity and excellent biocompatibility. Carbon dots have been widely applied in many areas such as bioimaging (W. Li, Zhang, Kong, Feng, Wang, Wang, et al., 2013; Yang, Cao, Luo, Lu, Wang, Wang, et al., 2009), catalysis (Zhuo, Shao, & Lee, 2012) and sensors. The chemical sensing systems based on CDs are mainly used for the detections of metal ions and related anions (Lim, Shen, & Gao, 2015; Lin, Rong, Luo, Chen, Wang, & Chen, 2014). In this paper, we prepared carbon dots (F-CDs) by simple hydrothermal reaction of feather in the presence of H2O2 and ammonia. The fluorescence of F-CDs can be selectively quenched by Cu2+ ion. Based this property, we constructed one kind of 3
off-on-off sensor for organophosphorus pesticides. This sensor was based on the following principles: (1) AChE can catalyze acetylthiocholine chloride (ATChCl) to form TCh, which can react with Cu 2+ ion and restore the fluorescence of F-CDs; (2) OPs can inhibit the activity of acetyl cholinesterase (AChE), which can turn the fluorescence of F-CDs off again. As an example of organophosphorus pesticides, dichlorvos was detected. Experimental results showed that the enzyme inhibition rate was proportional to the logarithm of the dichlorvos concentration in the range 6.0 × 10 -9 to 6.0 × 10-8 M with the detection limit (S/N = 3) of 3.8 × 10-9 M. This determination method showed a low detection limit, good selectivity and high reproducibility. This sensing system has been successfully used for the analysis of cabbage and fruit juice samples. EXPERIMENTAL SECTION 2.1. Materials Acetylcholinesterase (EC 3.1.1.7, from electrophorus electricus, lyophilized powder), acetylthiocholine chloride and OPs were purchased from Sigma-Aldrich (USA). NaH2PO4 and Na2HPO4 were purchased from Aladdin Ltd. (Shanghai, China). All chemicals were used as received without further purification. The water used throughout all the experiments was purified through redistillation process. The feather was obtained from a local poultry market. The cabbage and fruit juice samples were from a local supermarket. 2.2. Synthesis of F-CDs
4
Feather (0.5 g), 10 mL H2O2 (30%) and 10 mL ammonia (25-28%) were mixed, then added into a 40 mL Teflon-lined autoclave and heated at 160 ℃ for 7 h. After cooling to room temperature, the resulting brown solution was centrifuged at 3000 rpm for 15 min to remove the large dots, and was extensively dialyzed against redistilled water in a dialysis membrane (2000 MW CO) for 3 d. By freeze-dried method, brown powder was obtained for further characterization. The yield was calculated to be ca. 2.4%. The resulting powder was dispersed in distilled water at a concentration of 0.5 mg/mL for further use. 2.3. Characterization Transmission electron microscopy (TEM) and high resolution transmission electron microscopy (HRTEM) were obtained using Tecnai G2 F20 (USA, 200kV). Fourier transform infrared spectra (FTIR) were obtained on a Thermo Nicolet-380 IR spectrophotometer (USA). X-ray photoelectron spectroscopy (XPS) data was collected using Al Kα excitation source (1486.6 eV) in a Thermo ESCALAB 250XI apparatus (USA). Fluorescence spectra were obtained with a Cary Eclipse spectrophotometer (USA, VARIAN). UV-vis absorption spectrum data were collected using a UV-2450 Shimadzu Vis-spectrometer (Japan). X-ray powder diffraction (XRD) experiment were performed using a Rigaku DLMAX-2550 V diffractometer (40 kV, Cu Ka (k = 1.54056 Ǻ); scan speed of 6º/min). Zeta potential was obtained using Malvern Nano ZS90 Zetasizer (UK). 2.4. Cu2+ quenching assay procedure 100 µL 0.18 mg/mL F-CDs solution, 895 µL 0.2 M Phosphate Buffer (PBS) and 5
5 µL of different concentration of CuSO4 were mixed and equilibrated for 5 min at room temperature. The fluorescence spectra of the system were recorded under excitation at 340 nm. 2.5. Detection of DDVP in phosphate buffer The procedure for DDVP determination was as follows: (1) 100 µL AChE was first incubated with 4.90 mL 0.2 M PBS containing different concentrations of DDVP at certain temperature for a period of time; (2) 1.00 mL ATChCl, 1.00 mL CuSO4 (0.10 mM) and 3.00 mL F-CDs (0.06 mg/mL) were added into the above solution and the fluorescence signal was monitored over time. The inhibition rate of DDVP (I%) was adopted as a signal for the detection of DDVP. I% was calculated using eqn (1),
I% =
FAChE − FDDVP × 100% FAChE − FCu (II)
(1)
where FCu(II) was the fluorescence intensity after Cu2+ ion quenching experiment, FAChE stood for the fluorescence intensity after the recovery experiment of AChE, FDDVP was the fluorescence intensity after the DDVP inhibition reaction and AChE recovery experiment. 2.6. Measurement and calibration of DDVP in cabbage and fruit juice The pretreatment of cabbage was on the basis of previous report with modification (Zheng, Li, Dai, Liu, & Tang, 2011). 0.2 kg of cabbage was spiked with different quantities of DDVP. After overnight, the cabbage sample was first chopped and extracted with 20 mL PBS 8.0. For fruit juice samples, first, different quantities of DDVP were added into the solution, and then the solution was filtered through a 6
0.22-µm membrane, and mixed with an equal volume of 0.2 M PBS (pH = 8.0) to ensure the pH value of the solution. DDVP determination procedure in these samples was described as follows: (1) 10 µL 5 U/mL AChE was added into 490 µL sample solution and incubated at 35 ℃ for 10 min; (2) 100 µL 0.6 mM ATChCl, 100 µL 0.10 mM CuSO4 and 300 µL 0.06 mg/mL F-CDs were added into the above solution, after 3 min, the fluorescence spectra were recorded under excitation at 340 nm. The results were compared with the calibration curve, and the DDVP concentration could be obtained. 3. Results and discussion 3.1. Formation mechanism and characterization of F-CDs. Feather was most composed of keratin. Owing to the existence of plenty of disulfide bonds, keratin was insoluble at room temperature. Under the synergistic interaction of H2O2 and ammonia, keratin can be degraded into amino acids. H2O2 can oxidize disulfide bonds to sulfonic groups, which resulted in the degradation of keratin (Yuen, Kan, & Cheng, 2007). Ammonia can break the peptide bonds of keratin, and facilitate the dissolution of keratin (Gousterova, Braikova, Goshev, Christov, Tishinov, Vasileva-Tonkova, et al., 2005). Under hydrothermal reaction and oxidization action of H2O2, the carbonization reaction of amino acids can take place to form carbon dots. From the TEM images (Fig. 1b and 1c), we can see that the F-CDs were well-dispersed spherical dots with average diameters of 3.2 nm (based on statistical analysis of more than 200 dots). The inset of Fig. 1b showed these nanodots 7
were mainly distributed in the range of 2-4 nm. In our previous report (Hou, Li, Sun, Ai, & Wang, 2014), carbon nanospheres with average diameters of 49 nm were obtained by the hydrothermal reaction of hair in water without any additive. These two kinds of carbon dots were significantly different in morphology. The size of F-CDs was smaller and more uniform. The HRTEM image of F-CDs in Fig. 1d showed the lattice spacing (0.24 nm) of the (100) facet in graphite (Hu, Niu, Sun, Yang, Zhao, & Du, 2009; Lu, Yang, Wang, Lim, Wang, & Loh, 2009; Qin, Lu, Asiri, Al-Youbi, & Sun, 2013). The XRD pattern of F-CDs (Fig. 1e) showed a broad peak centered at 20.1°(0.44 nm), which may be attributed to the interlayer spacing (0.34 nm) of the (002) facet in graphite. The enlargement of interlayer spacing may be attributed to the presence of nitrogen and oxygen containing groups. Fig. S1 showed the FTIR spectrum of the F-CDs. The peaks around 3419 and 3292 cm-1 can be attributed to the stretching vibration of O–H and N–H (J. Jiang, He, Li, & Cui, 2012). The peaks around 1630 cm-1 were assigned to the bending vibration of C=O and N–H (J. Jiang, He, Li, & Cui, 2012). X-ray photoelectron spectroscopy of the F-CDs was shown in Fig. S2. The spectrum of C1s (Fig. S2b) showed that carbon atoms presented in five different form, corresponding to C=C, C–C, C–N, C–O and C=O (S. Liu, Tian, Wang, Zhang, Qin, Luo, et al., 2012). The spectrum of O1s (Fig. S2c) confirmed the presence of two oxygen states of C=O and C–O (S. Liu, et al., 2012). The N1s spectrum (Fig. S2d) revealed two nitrogen species of C–N–C and N–H (W. Li, et al., 2013). The spectrum of S2p (Fig. S2e) can be deconvoluted into several single peaks that corresponded to –SO2–, –SO3– and –SO4– 8
functional groups (Sun, Ban, Zhang, Wu, Zhang, & Zhu, 2013). The content of S was as low as 0.84%, which was much lower than that of feather. This phenomenon indicated that most of S element was oxidized to form sulfonic compounds. These results of FTIR and XPS indicated that there were carboxylic acid, amino and other oxygen-containing or nitrogen-containing functional groups in the F-CDs. Due to the presence of abundant hydrophilic groups on the surface of the F-CDs, the F-CDs possessed high stability in water with a zeta potential of -41.3 mV. The F-CDs had a weak UV-vis absorption around ca. 275 nm (Fig. S3b), which was ascribed to the π–π* transition of C=C (Y. Li, Zhao, Cheng, Hu, Shi, Dai, et al., 2011; Zhu, Zhai, & Dong, 2012). The photoluminescence (PL) emission spectra of the F-CDs at different excitation wavelengths were shown in Fig. S3b. When the PL excitation wavelength changed from 300 to 400 nm, the PL emission peak correspondingly shifted from 409 to 455 nm. Solvent dependent fluorescence behavior was observed in most carbon-based materials (Bao, Zhang, Tian, Zhang, Liu, Lin, et al., 2011; Fang, Guo, Li, Zhu, Ren, Dong, et al., 2011; Jeong, Cho, Lim, Song, & Chung, 2009; R. Liu, Wu, Liu, Koynov, Knoll, & Li, 2009; Loh, Bao, Eda, & Chhowalla, 2010; X. Wang, Cao, Yang, Lu, Meziani, Tian, et al., 2010; Welsher, Liu, Sherlock, Robinson, Chen, Daranciang, et al., 2009). In the following experiments, the excitation wavelength was fixed at 340 nm owing to the strongest fluorescence emission. The quantum yield of F-CDs using quinine sulfate as standards was 11.6% at 340 nm excitation. Fig. S3c showed that the PL emission spectra of the F-CDs in 9
water, dimethylformamide (DMF), tetrahydrofuran (THF) and acetone at 340 nm excitation. The PL spectra showed different emission wavelengths in different solvent. This solvent effect could be ascribed to solvent attachment or the formation of different emissive traps on the surfaces of the F-CDs (Pan, Zhang, Li, Wu, Yan, & Wu, 2010; Paraknowitsch, Zhang, Wienert, & Thomas, 2013; Sun, Ban, Zhang, Wu, Zhang, & Zhu, 2013). The effects of the pH and ionic strength of solutions, and UV exposure on the fluorescence intensity of F-CDs were studied to discuss the PL stability of F-CDs (Fig. S4). From Fig. S4a, we can see that the fluorescence intensity of F-CDs changed less at pH 7–9, which was in favour of construction of enzyme sensor. Fig. S4b showed that there was no obvious change in PL intensity at different ionic strengths. Fig. S4c indicated that F-CDs had an excellent photostability. 3.2. The quenching effect of Cu2+ ion on the fluorescence of F-CDs Fig. 2a showed the PL spectra of F-CDs containing different concentrations of Cu2+ ion. From Fig. 2a, we can see that Cu 2+ ion had intense quenching effect on the fluorescence of F-CDs. Under the same conditions, the quenching experiments of other metal ions were carried out, including Hg2+, Ag+, Ni2+, Co2+, Mg2+, Mn2+, Zn2+, Ca2+, Fe3+, Pb2+ and Cd2+ ions (Fig. 2b, I0 and I stand for the PL intensity of F-CDs before and after quenching experiment, respectively). The results showed that other ions had no obvious quenching effect on the fluorescence of F-CDs. Fig. 2c-d showed the effect of pH and salting strength on the fluorescence quenching of Cu 2+ ion. From Fig. 2c, we can see that pH can obviously influence the quenching efficiency. In the 10
pH 2 PBS, the quenching efficiency of Cu 2+ ion was quite low. The quenching efficiency of Cu 2+ ion at pH 7-9 was higher and changed less with pH, which would benefit the determination of OPs. The quenching effect of Cu2+ ion was mainly attributed to the charge transfer between F-CDs and Cu2+ ion. This phenomenon can be attributed to the form change of groups on the surface of F-CDs with pH. The zeta potential determination can prove this viewpoint. In PBS 2 buffer, the zeta potential was close to zero, owing to the protonation of carboxy groups. However, in PBS 8 buffer, the zeta potential was -42.8 mV. Fig. 2d indicated that the quenching efficiency in solutions with different salting strength made no difference, so no extra salts were added in the following experiments. 3.3. Detection principle of OPs sensor Fig. 3a illustrated the fluorescence switch mechanism for the detection of OPs based on F-CDs and Cu2+ ion system. The sensor was based on the following principles: (1) AChE can catalyze acetylthiocholine chloride to form TCh; (2) a redox reaction between Cu 2+ ion and TCh can take place to restore the fluorescence of carbon dots; (2) OPs can inhibit the activity of acetylcholinesterase and inhibit the recovery of fluorescence. From Fig. 3b, we can see that when AChE and ATChCl were introduced into the F-CDs and Cu2+ ion system, the PL intensity was recovered at large extent. However, when AChE was first incubated with dichlorvos, the recovery of fluorescence was inhibited. We have carried out XPS characterization about the products of F-CDs/Cu2+/AChE/ATChCl to study the mechanism of the
11
reaction between TCh and Cu 2+ ion. Fig. 3c showed the spectrum of Cu2p, the spectrum contained two obvious peaks that corresponded to Cu2p3/2 (932.8 eV) and Cu2p1/2 (952.5 eV) (Ghodselahi, Vesaghi, Shafiekhani, Baghizadeh, & Lameii, 2008). The satellite peak of Cu2p3/2 was not found, which demonstrated the absence of Cu 2+ ion. So, the reaction mechanism can be as following: a redox reaction between Cu 2+ ion and TCh can take place to form Cu(I)−TCh complex and the oxidized TCh. 3.4. Optimization of the experimental conditions for DDVP determination As a kind of organphosphorus pesticides, dichlorvos was widely used as in agricultural production. So, we chose dichlorvos as the inhibitor of AChE. As a kind of sensor based on the activity of enzymes, many factors should be optimized to obtain high sensitivity, such as the pH of solution, the concentration of enzyme, the concentration of substrate, the reaction temperature and the incubation time of AChE with OPs. In optimization test, the concentration of DDVP was fixed at 4.0 × 10-8 M. The inhibition rate of DDVP (I%) was adopted as a signal for the detection of DDVP. DDVP can be hydrolyzed at basic solution. The optimum pH for the activity of AChE is 8.0-9.0. In comprehensive consideration of the stability of DDVP and the activity of AChE, the pH was fixed at 8.0 in the following experiments. By initial experiments, the incubation time of AChE with OPs was fixed at 10 min, and the reaction temperature was fixed at 35 ℃. As one kind of sensor based on the activity of enzymes, the reaction time in the determination experiment was strictly controlled. When the concentration of AChE was 0.100 U/mL and the concentration of 12
ATChCl was 100 µM, the inhibition rates of different reaction time were obtained (Fig. 4a). From the Fig. 4a, we can see that when the reaction time were 2, 3 and 4 min, the inhibition rates were higher. These three times were chosen in the final optimum experiments. When the concentration of ATChCl was 100 µM and the reaction time was 3 min, the inhibition rates of different concentrations of AChE were obtained (Fig. 4b). Fig. 4b exhibited that the appropriate concentrations of AChE were 0.030, 0.040, and 0.050 U/mL. These three concentrations of AChE were chosen in the final optimum experiments. When the concentration of AChE was 0.040 U/mL and the reaction time was 3 min, the inhibition rates of the different concentrations of ATChCl were shown in Fig. 4c. The Fig. 4c indicated that the appropriate concentrations of ATChCl were 50, 60 and 70 µM. These three concentrations of ATChCl were chosen in the final optimum experiments. Owing to the chosen optimum experimental conditions, the final optimum experiments were carried out (Table S1). When the concentrations of AChE was 0.050 U/mL, the concentration of ATChCl was 60 µM, and the reaction time was 3 min, the highest inhibition rate (75.24%) was obtained. The optimal determination conditions were as follows: first, 100 µL 5 U/mL AChE was first incubated with 4.90 mL 0.2 M PBS containing different concentrations of DDVP in pH 8 PBS at 35 ℃ for 10 min; second, 1.00 mL 0.6 mM ATChCl, 1.00 mL 0.10 mM CuSO4 and 3.00 mL 0.06 mg/mL F-CDs were added into the above solution, and the mixed solution reacted at 35 ℃ for 3 min; final, the PL spectrum of the mixed solution was recorded. 13
Fig. 5a showed fluorescence spectra of the F-CDs/Cu 2+/AChE/ATChCl system after incubation with different concentrations of DDVP. Fig. 5b showed the relationship between I% and log CDDVP. The insert of Fig. 5b exhibited a good linear relationship between the inhibition rate and log CDDVP in the range of 6.0 × 10-9 – 6.0 × 10 -8 M. The semi-log dependence of inhibition rate on the OPs concentration has been found in previous reported OPs sensors based on enzyme inhibition mechanism (Gao, Tang, & Su, 2012; Liang, Fan, Pan, Jiang, Wang, Yang, et al., 2012; Zheng, Li, Dai, Liu, & Tang, 2011). The regression equation was I (%) = 706.97 + 85.27 log CDDVP (R2 = 0.998). Under the same conditions, the detections of malathion and ethion were carried out. From Fig. S5, we can see that the regression equation of malathion was I (%) = 706.97 + 85.27 log Cmalathion (R2 = 0.996) in the range of 6.0 × 10-9 – 8.0 × 10-8 M, and the regression equation of ethion was I (%) = 677.15 + 81.40 log Cethion (R2 = 0.997) in the range of 8.0 × 10-9 – 8.0 × 10 -8 M. The detection limits for DDVP, malathion and ethion at a signal-to-noise ratio of 3 were estimated to be 3.8 × 10-9 M (8.4 × 10-4 ppm), 3.4 × 10-9 M (1.1 × 10-3 ppm) and 4.2 × 10-9 M (1.6 × 10-3 ppm), respectively, which were much lower than the maximum residue limits (MRLs) as reported in the European Union pesticides database as well as those from the U.S. Department Agriculture (MRLs are 0.01, 0.02 and 0.01 ppm for DDVP, malathion and ethion, respectively). These results suggested that this sensing system could be applied in the determination of different kinds of OPs. Table S2 listed different fluorescent probes for OPs detection, suggesting the sensitivity of this method was higher than that of 14
other fluorescent sensors. Furthermore, the reagents of this method were green and economical, for example, feather, H2O2, ammonia and CuSO4. 3.5. The anti-interference ability, stability and reproducibility To evaluate the selectivity of the proposed method, coexistence substances were added into the DDVP solution. The interference effect of 106-fold for NaCl and KCl, 800-fold for CaCl2 and Zn(Ac)2, 200-fold for FeCl3 and ascorbic acid, 104-fold for glucose, sodium acetate and sodium citrate, 200-fold for glycerol on the determination of 1.0 × 10 -8 M DDVP were studied (Fig. S6). The results showed that these coexistence substances had no influence on the signals of 1.0 × 10 -8 M DDVP with deviations below 5%. These results showed this method displayed good selectivity. To evaluate the stability of this sensor system, the prepared F-CDs was first kept in refrigerator at 4 ℃ for 30 days, then used in the determination of 1.0 × 10-8 M DDVP. The determination value was 98.8% of its initial response, which indicated that the method had good stability. To evaluate the reproducibility of this method, five parallel prepared F-CDs were using in the determination of 1.0 × 10-8 M DDVP. The relative standard deviation was 5.2%, which suggested that the sensor displayed good reproducibility. 3.6. Determination of DDVP in the cabbage and fruit juice samples In order to further investigate the accuracy, selectivity and repeatability of the proposed method, different concentrations of DDVP in the cabbage and fruit juice samples were determined (Table 1). From Table 1, it can be seen that the recoveries of 15
DDVP in these samples were in the range 94–104%, and the RSDs were less than 7.0%. The results revealed that this method was applicable in the determination of OPs in organophosphate compound-contaminated food samples. 4. Conclusion In conclusion, we have constructed one kind of fluorescence sensor based on CDs for the determination of DDVP. Both the preparation method and detection process were simple, green and economic. These carbon dots were obtained by hydrothermal reaction of feather in presence of H2O2 and ammonia. The sensing system was based on the selective fluorescence quench effects of Cu 2+ ion. This sensing system has been successfully used for the analysis of cabbage and fruit juice samples with high selectivity and sensitivity. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 21375079, 51402175) and Project of Development of Science and Technology of Shandong Province, China (No. 2013GZX20109). References Amini, N., Shariatgorji, M., Crescenzi, C., & Thorsén, G. (2009). Screening and quantification of pesticides in water using a dual-function graphitized carbon black disk. Analytical Chemistry, 82(1), 290-296. Azab, H. A., Duerkop, A., Anwar, Z., Hussein, B. H., Rizk, M. A., & Amin, T. (2013). Luminescence recognition of different organophosphorus pesticides by the luminescent Eu (III)–pyridine-2, 6-dicarboxylic acid probe. Analytica Chimica Acta, 759, 81-91. Bao, L., Zhang, Z. L., Tian, Z. Q., Zhang, L., Liu, C., Lin, Y., Qi, B., & Pang, D. W. (2011). Electrochemical tuning of luminescent carbon nanodots: from preparation to luminescence mechanism. Advanced Materials, 23(48), 5801-5806. Fang, Y., Guo, S., Li, D., Zhu, C., Ren, W., Dong, S., & Wang, E. (2011). Easy synthesis and imaging applications of cross-linked green fluorescent hollow carbon nanoparticles. ACS Nano, 6(1), 400-409. 16
S0308-8146(15)30268-5 http://dx.doi.org/10.1016/j.foodchem.2015.11.134 FOCH 18475
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
28 July 2015 2 November 2015 28 November 2015
Please cite this article as: Hou, J., Dong, G., Tian, Z., Lu, J., Wang, Q., Ai, S., Wang, M., A sensitive fluorescent sensor for selective determination of dichlorvos based on the recovered fluorescence of carbon dots-Cu(II) system, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/j.foodchem.2015.11.134
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A sensitive fluorescent sensor for selective determination of dichlorvos based on the recovered fluorescence of carbon dots-Cu(II) system Juying Hou a,b, Guangjuan Donga, Zhengbin Tiana, Jiutian Lua, Qianqian Wanga, Shiyun Ai*,a, Minglin Wang*,b a
College of Chemistry and Material Science, Shandong Agricultural University,
Taian, Shandong 271018, P.R. China b
College of Food Science and Engineering, Shandong Agricultural University, Taian,
Shandong 271018, P.R. China
*
Corresponding authors:
Tel: +86 538 8247660 Fax: +86 538 8242251 E-mail address: [email protected] (S.Y. Ai) [email protected] (M.L. Wang) 1
Abstract In this paper, a simple and sensitive fluorescent sensor for dichlorvos was first constructed based on carbon dots-Cu(II) system. These carbon dots were obtained by simple hydrothermal reaction of feather. The fluorescence of these carbon dots can be selectively quenched by Cu2+ ion. When acetylcholinesterase and acetylthiocholine were introduced into the system, thiocholine came into being, which can react with Cu2+ ion and restore the fluorescence of the system. The reaction mechanism between Cu2+ ion and thiocholine was confirmed by X-ray photoelectron spectroscopy. As one kind of acetylcholinesterase inhibitor, organophosphorus pesticides can be detected based on this sensing system. As an example of organophosphorus pesticides, dichlorvos was detected with a linear range of 6.0 × 10-9 - 6.0 × 10 -8 M. This sensing system has been successfully used for the analysis of cabbage and fruit juice samples. Keywords: Carbon dots; Fluorescent; Sensor; Organophosphorus compounds; Acetylcholinesterase; Enzyme inhibition 1. Introduction Organophosphorus compounds (OPs) have been extensively used as pesticides in modern agriculture. As estimated, millions of tons of organophosphate pesticides are used in agriculture each year, which can raise serious human health and environmental concerns. More than two million people die from OPs poisoning every year. Therefore, there is an urgent demand for the determination of OPs. Conventional analytical techniques, such as liquid chromatography, gas chromatography and tandem mass spectrometer (Amini, Shariatgorji, Crescenzi, & Thorsén, 2009; Lambropoulou & Albanis, 2007; Zhou, Xiao, & Li, 2012) need expensive 2
equipments, skilled personnel and time-consuming treatment of samples. Owing to high sensitivity, low-cost, and operational rapidity and simplicity, fluorescent detection have attracted tremendous attention (Azab, Duerkop, Anwar, Hussein, Rizk, & Amin, 2013; Gao, Tang, & Su, 2012; Long, Li, Zhang, & Yao, 2015; Meng, Wei, Ren, Ren, & Tang, 2013; K. Zhang, Yu, Liu, Sun, Yu, Liu, et al., 2014). However, fluorescence materials using in these methods suffered from some disadvantages, such as tedious preparation procedures, poisonous synthetic conditions, and toxicity of material etc. Recently, fluorescent carbon dots (CDs) have attracted tremendous research interests (K. Jiang, Sun, Zhang, Lu, Wu, Cai, et al., 2015; J. Wang, Zhang, Huang, Liu, Leung, & Wáng, 2015; Xu, Pu, Zhao, Dong, Gao, Chen, et al., 2015; L. Zhang, Han, Zhu, Zhai, & Dong, 2015), owing to their appealing advantages, such as good water-solubility, high chemical inertness, good photostability, low cytotoxicity and excellent biocompatibility. Carbon dots have been widely applied in many areas such as bioimaging (W. Li, Zhang, Kong, Feng, Wang, Wang, et al., 2013; Yang, Cao, Luo, Lu, Wang, Wang, et al., 2009), catalysis (Zhuo, Shao, & Lee, 2012) and sensors. The chemical sensing systems based on CDs are mainly used for the detections of metal ions and related anions (Lim, Shen, & Gao, 2015; Lin, Rong, Luo, Chen, Wang, & Chen, 2014). In this paper, we prepared carbon dots (F-CDs) by simple hydrothermal reaction of feather in the presence of H2O2 and ammonia. The fluorescence of F-CDs can be selectively quenched by Cu2+ ion. Based this property, we constructed one kind of 3
off-on-off sensor for organophosphorus pesticides. This sensor was based on the following principles: (1) AChE can catalyze acetylthiocholine chloride (ATChCl) to form TCh, which can react with Cu 2+ ion and restore the fluorescence of F-CDs; (2) OPs can inhibit the activity of acetyl cholinesterase (AChE), which can turn the fluorescence of F-CDs off again. As an example of organophosphorus pesticides, dichlorvos was detected. Experimental results showed that the enzyme inhibition rate was proportional to the logarithm of the dichlorvos concentration in the range 6.0 × 10 -9 to 6.0 × 10-8 M with the detection limit (S/N = 3) of 3.8 × 10-9 M. This determination method showed a low detection limit, good selectivity and high reproducibility. This sensing system has been successfully used for the analysis of cabbage and fruit juice samples. EXPERIMENTAL SECTION 2.1. Materials Acetylcholinesterase (EC 3.1.1.7, from electrophorus electricus, lyophilized powder), acetylthiocholine chloride and OPs were purchased from Sigma-Aldrich (USA). NaH2PO4 and Na2HPO4 were purchased from Aladdin Ltd. (Shanghai, China). All chemicals were used as received without further purification. The water used throughout all the experiments was purified through redistillation process. The feather was obtained from a local poultry market. The cabbage and fruit juice samples were from a local supermarket. 2.2. Synthesis of F-CDs
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Feather (0.5 g), 10 mL H2O2 (30%) and 10 mL ammonia (25-28%) were mixed, then added into a 40 mL Teflon-lined autoclave and heated at 160 ℃ for 7 h. After cooling to room temperature, the resulting brown solution was centrifuged at 3000 rpm for 15 min to remove the large dots, and was extensively dialyzed against redistilled water in a dialysis membrane (2000 MW CO) for 3 d. By freeze-dried method, brown powder was obtained for further characterization. The yield was calculated to be ca. 2.4%. The resulting powder was dispersed in distilled water at a concentration of 0.5 mg/mL for further use. 2.3. Characterization Transmission electron microscopy (TEM) and high resolution transmission electron microscopy (HRTEM) were obtained using Tecnai G2 F20 (USA, 200kV). Fourier transform infrared spectra (FTIR) were obtained on a Thermo Nicolet-380 IR spectrophotometer (USA). X-ray photoelectron spectroscopy (XPS) data was collected using Al Kα excitation source (1486.6 eV) in a Thermo ESCALAB 250XI apparatus (USA). Fluorescence spectra were obtained with a Cary Eclipse spectrophotometer (USA, VARIAN). UV-vis absorption spectrum data were collected using a UV-2450 Shimadzu Vis-spectrometer (Japan). X-ray powder diffraction (XRD) experiment were performed using a Rigaku DLMAX-2550 V diffractometer (40 kV, Cu Ka (k = 1.54056 Ǻ); scan speed of 6º/min). Zeta potential was obtained using Malvern Nano ZS90 Zetasizer (UK). 2.4. Cu2+ quenching assay procedure 100 µL 0.18 mg/mL F-CDs solution, 895 µL 0.2 M Phosphate Buffer (PBS) and 5
5 µL of different concentration of CuSO4 were mixed and equilibrated for 5 min at room temperature. The fluorescence spectra of the system were recorded under excitation at 340 nm. 2.5. Detection of DDVP in phosphate buffer The procedure for DDVP determination was as follows: (1) 100 µL AChE was first incubated with 4.90 mL 0.2 M PBS containing different concentrations of DDVP at certain temperature for a period of time; (2) 1.00 mL ATChCl, 1.00 mL CuSO4 (0.10 mM) and 3.00 mL F-CDs (0.06 mg/mL) were added into the above solution and the fluorescence signal was monitored over time. The inhibition rate of DDVP (I%) was adopted as a signal for the detection of DDVP. I% was calculated using eqn (1),
I% =
FAChE − FDDVP × 100% FAChE − FCu (II)
(1)
where FCu(II) was the fluorescence intensity after Cu2+ ion quenching experiment, FAChE stood for the fluorescence intensity after the recovery experiment of AChE, FDDVP was the fluorescence intensity after the DDVP inhibition reaction and AChE recovery experiment. 2.6. Measurement and calibration of DDVP in cabbage and fruit juice The pretreatment of cabbage was on the basis of previous report with modification (Zheng, Li, Dai, Liu, & Tang, 2011). 0.2 kg of cabbage was spiked with different quantities of DDVP. After overnight, the cabbage sample was first chopped and extracted with 20 mL PBS 8.0. For fruit juice samples, first, different quantities of DDVP were added into the solution, and then the solution was filtered through a 6
0.22-µm membrane, and mixed with an equal volume of 0.2 M PBS (pH = 8.0) to ensure the pH value of the solution. DDVP determination procedure in these samples was described as follows: (1) 10 µL 5 U/mL AChE was added into 490 µL sample solution and incubated at 35 ℃ for 10 min; (2) 100 µL 0.6 mM ATChCl, 100 µL 0.10 mM CuSO4 and 300 µL 0.06 mg/mL F-CDs were added into the above solution, after 3 min, the fluorescence spectra were recorded under excitation at 340 nm. The results were compared with the calibration curve, and the DDVP concentration could be obtained. 3. Results and discussion 3.1. Formation mechanism and characterization of F-CDs. Feather was most composed of keratin. Owing to the existence of plenty of disulfide bonds, keratin was insoluble at room temperature. Under the synergistic interaction of H2O2 and ammonia, keratin can be degraded into amino acids. H2O2 can oxidize disulfide bonds to sulfonic groups, which resulted in the degradation of keratin (Yuen, Kan, & Cheng, 2007). Ammonia can break the peptide bonds of keratin, and facilitate the dissolution of keratin (Gousterova, Braikova, Goshev, Christov, Tishinov, Vasileva-Tonkova, et al., 2005). Under hydrothermal reaction and oxidization action of H2O2, the carbonization reaction of amino acids can take place to form carbon dots. From the TEM images (Fig. 1b and 1c), we can see that the F-CDs were well-dispersed spherical dots with average diameters of 3.2 nm (based on statistical analysis of more than 200 dots). The inset of Fig. 1b showed these nanodots 7
were mainly distributed in the range of 2-4 nm. In our previous report (Hou, Li, Sun, Ai, & Wang, 2014), carbon nanospheres with average diameters of 49 nm were obtained by the hydrothermal reaction of hair in water without any additive. These two kinds of carbon dots were significantly different in morphology. The size of F-CDs was smaller and more uniform. The HRTEM image of F-CDs in Fig. 1d showed the lattice spacing (0.24 nm) of the (100) facet in graphite (Hu, Niu, Sun, Yang, Zhao, & Du, 2009; Lu, Yang, Wang, Lim, Wang, & Loh, 2009; Qin, Lu, Asiri, Al-Youbi, & Sun, 2013). The XRD pattern of F-CDs (Fig. 1e) showed a broad peak centered at 20.1°(0.44 nm), which may be attributed to the interlayer spacing (0.34 nm) of the (002) facet in graphite. The enlargement of interlayer spacing may be attributed to the presence of nitrogen and oxygen containing groups. Fig. S1 showed the FTIR spectrum of the F-CDs. The peaks around 3419 and 3292 cm-1 can be attributed to the stretching vibration of O–H and N–H (J. Jiang, He, Li, & Cui, 2012). The peaks around 1630 cm-1 were assigned to the bending vibration of C=O and N–H (J. Jiang, He, Li, & Cui, 2012). X-ray photoelectron spectroscopy of the F-CDs was shown in Fig. S2. The spectrum of C1s (Fig. S2b) showed that carbon atoms presented in five different form, corresponding to C=C, C–C, C–N, C–O and C=O (S. Liu, Tian, Wang, Zhang, Qin, Luo, et al., 2012). The spectrum of O1s (Fig. S2c) confirmed the presence of two oxygen states of C=O and C–O (S. Liu, et al., 2012). The N1s spectrum (Fig. S2d) revealed two nitrogen species of C–N–C and N–H (W. Li, et al., 2013). The spectrum of S2p (Fig. S2e) can be deconvoluted into several single peaks that corresponded to –SO2–, –SO3– and –SO4– 8
functional groups (Sun, Ban, Zhang, Wu, Zhang, & Zhu, 2013). The content of S was as low as 0.84%, which was much lower than that of feather. This phenomenon indicated that most of S element was oxidized to form sulfonic compounds. These results of FTIR and XPS indicated that there were carboxylic acid, amino and other oxygen-containing or nitrogen-containing functional groups in the F-CDs. Due to the presence of abundant hydrophilic groups on the surface of the F-CDs, the F-CDs possessed high stability in water with a zeta potential of -41.3 mV. The F-CDs had a weak UV-vis absorption around ca. 275 nm (Fig. S3b), which was ascribed to the π–π* transition of C=C (Y. Li, Zhao, Cheng, Hu, Shi, Dai, et al., 2011; Zhu, Zhai, & Dong, 2012). The photoluminescence (PL) emission spectra of the F-CDs at different excitation wavelengths were shown in Fig. S3b. When the PL excitation wavelength changed from 300 to 400 nm, the PL emission peak correspondingly shifted from 409 to 455 nm. Solvent dependent fluorescence behavior was observed in most carbon-based materials (Bao, Zhang, Tian, Zhang, Liu, Lin, et al., 2011; Fang, Guo, Li, Zhu, Ren, Dong, et al., 2011; Jeong, Cho, Lim, Song, & Chung, 2009; R. Liu, Wu, Liu, Koynov, Knoll, & Li, 2009; Loh, Bao, Eda, & Chhowalla, 2010; X. Wang, Cao, Yang, Lu, Meziani, Tian, et al., 2010; Welsher, Liu, Sherlock, Robinson, Chen, Daranciang, et al., 2009). In the following experiments, the excitation wavelength was fixed at 340 nm owing to the strongest fluorescence emission. The quantum yield of F-CDs using quinine sulfate as standards was 11.6% at 340 nm excitation. Fig. S3c showed that the PL emission spectra of the F-CDs in 9
water, dimethylformamide (DMF), tetrahydrofuran (THF) and acetone at 340 nm excitation. The PL spectra showed different emission wavelengths in different solvent. This solvent effect could be ascribed to solvent attachment or the formation of different emissive traps on the surfaces of the F-CDs (Pan, Zhang, Li, Wu, Yan, & Wu, 2010; Paraknowitsch, Zhang, Wienert, & Thomas, 2013; Sun, Ban, Zhang, Wu, Zhang, & Zhu, 2013). The effects of the pH and ionic strength of solutions, and UV exposure on the fluorescence intensity of F-CDs were studied to discuss the PL stability of F-CDs (Fig. S4). From Fig. S4a, we can see that the fluorescence intensity of F-CDs changed less at pH 7–9, which was in favour of construction of enzyme sensor. Fig. S4b showed that there was no obvious change in PL intensity at different ionic strengths. Fig. S4c indicated that F-CDs had an excellent photostability. 3.2. The quenching effect of Cu2+ ion on the fluorescence of F-CDs Fig. 2a showed the PL spectra of F-CDs containing different concentrations of Cu2+ ion. From Fig. 2a, we can see that Cu 2+ ion had intense quenching effect on the fluorescence of F-CDs. Under the same conditions, the quenching experiments of other metal ions were carried out, including Hg2+, Ag+, Ni2+, Co2+, Mg2+, Mn2+, Zn2+, Ca2+, Fe3+, Pb2+ and Cd2+ ions (Fig. 2b, I0 and I stand for the PL intensity of F-CDs before and after quenching experiment, respectively). The results showed that other ions had no obvious quenching effect on the fluorescence of F-CDs. Fig. 2c-d showed the effect of pH and salting strength on the fluorescence quenching of Cu 2+ ion. From Fig. 2c, we can see that pH can obviously influence the quenching efficiency. In the 10
pH 2 PBS, the quenching efficiency of Cu 2+ ion was quite low. The quenching efficiency of Cu 2+ ion at pH 7-9 was higher and changed less with pH, which would benefit the determination of OPs. The quenching effect of Cu2+ ion was mainly attributed to the charge transfer between F-CDs and Cu2+ ion. This phenomenon can be attributed to the form change of groups on the surface of F-CDs with pH. The zeta potential determination can prove this viewpoint. In PBS 2 buffer, the zeta potential was close to zero, owing to the protonation of carboxy groups. However, in PBS 8 buffer, the zeta potential was -42.8 mV. Fig. 2d indicated that the quenching efficiency in solutions with different salting strength made no difference, so no extra salts were added in the following experiments. 3.3. Detection principle of OPs sensor Fig. 3a illustrated the fluorescence switch mechanism for the detection of OPs based on F-CDs and Cu2+ ion system. The sensor was based on the following principles: (1) AChE can catalyze acetylthiocholine chloride to form TCh; (2) a redox reaction between Cu 2+ ion and TCh can take place to restore the fluorescence of carbon dots; (2) OPs can inhibit the activity of acetylcholinesterase and inhibit the recovery of fluorescence. From Fig. 3b, we can see that when AChE and ATChCl were introduced into the F-CDs and Cu2+ ion system, the PL intensity was recovered at large extent. However, when AChE was first incubated with dichlorvos, the recovery of fluorescence was inhibited. We have carried out XPS characterization about the products of F-CDs/Cu2+/AChE/ATChCl to study the mechanism of the
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reaction between TCh and Cu 2+ ion. Fig. 3c showed the spectrum of Cu2p, the spectrum contained two obvious peaks that corresponded to Cu2p3/2 (932.8 eV) and Cu2p1/2 (952.5 eV) (Ghodselahi, Vesaghi, Shafiekhani, Baghizadeh, & Lameii, 2008). The satellite peak of Cu2p3/2 was not found, which demonstrated the absence of Cu 2+ ion. So, the reaction mechanism can be as following: a redox reaction between Cu 2+ ion and TCh can take place to form Cu(I)−TCh complex and the oxidized TCh. 3.4. Optimization of the experimental conditions for DDVP determination As a kind of organphosphorus pesticides, dichlorvos was widely used as in agricultural production. So, we chose dichlorvos as the inhibitor of AChE. As a kind of sensor based on the activity of enzymes, many factors should be optimized to obtain high sensitivity, such as the pH of solution, the concentration of enzyme, the concentration of substrate, the reaction temperature and the incubation time of AChE with OPs. In optimization test, the concentration of DDVP was fixed at 4.0 × 10-8 M. The inhibition rate of DDVP (I%) was adopted as a signal for the detection of DDVP. DDVP can be hydrolyzed at basic solution. The optimum pH for the activity of AChE is 8.0-9.0. In comprehensive consideration of the stability of DDVP and the activity of AChE, the pH was fixed at 8.0 in the following experiments. By initial experiments, the incubation time of AChE with OPs was fixed at 10 min, and the reaction temperature was fixed at 35 ℃. As one kind of sensor based on the activity of enzymes, the reaction time in the determination experiment was strictly controlled. When the concentration of AChE was 0.100 U/mL and the concentration of 12
ATChCl was 100 µM, the inhibition rates of different reaction time were obtained (Fig. 4a). From the Fig. 4a, we can see that when the reaction time were 2, 3 and 4 min, the inhibition rates were higher. These three times were chosen in the final optimum experiments. When the concentration of ATChCl was 100 µM and the reaction time was 3 min, the inhibition rates of different concentrations of AChE were obtained (Fig. 4b). Fig. 4b exhibited that the appropriate concentrations of AChE were 0.030, 0.040, and 0.050 U/mL. These three concentrations of AChE were chosen in the final optimum experiments. When the concentration of AChE was 0.040 U/mL and the reaction time was 3 min, the inhibition rates of the different concentrations of ATChCl were shown in Fig. 4c. The Fig. 4c indicated that the appropriate concentrations of ATChCl were 50, 60 and 70 µM. These three concentrations of ATChCl were chosen in the final optimum experiments. Owing to the chosen optimum experimental conditions, the final optimum experiments were carried out (Table S1). When the concentrations of AChE was 0.050 U/mL, the concentration of ATChCl was 60 µM, and the reaction time was 3 min, the highest inhibition rate (75.24%) was obtained. The optimal determination conditions were as follows: first, 100 µL 5 U/mL AChE was first incubated with 4.90 mL 0.2 M PBS containing different concentrations of DDVP in pH 8 PBS at 35 ℃ for 10 min; second, 1.00 mL 0.6 mM ATChCl, 1.00 mL 0.10 mM CuSO4 and 3.00 mL 0.06 mg/mL F-CDs were added into the above solution, and the mixed solution reacted at 35 ℃ for 3 min; final, the PL spectrum of the mixed solution was recorded. 13
Fig. 5a showed fluorescence spectra of the F-CDs/Cu 2+/AChE/ATChCl system after incubation with different concentrations of DDVP. Fig. 5b showed the relationship between I% and log CDDVP. The insert of Fig. 5b exhibited a good linear relationship between the inhibition rate and log CDDVP in the range of 6.0 × 10-9 – 6.0 × 10 -8 M. The semi-log dependence of inhibition rate on the OPs concentration has been found in previous reported OPs sensors based on enzyme inhibition mechanism (Gao, Tang, & Su, 2012; Liang, Fan, Pan, Jiang, Wang, Yang, et al., 2012; Zheng, Li, Dai, Liu, & Tang, 2011). The regression equation was I (%) = 706.97 + 85.27 log CDDVP (R2 = 0.998). Under the same conditions, the detections of malathion and ethion were carried out. From Fig. S5, we can see that the regression equation of malathion was I (%) = 706.97 + 85.27 log Cmalathion (R2 = 0.996) in the range of 6.0 × 10-9 – 8.0 × 10-8 M, and the regression equation of ethion was I (%) = 677.15 + 81.40 log Cethion (R2 = 0.997) in the range of 8.0 × 10-9 – 8.0 × 10 -8 M. The detection limits for DDVP, malathion and ethion at a signal-to-noise ratio of 3 were estimated to be 3.8 × 10-9 M (8.4 × 10-4 ppm), 3.4 × 10-9 M (1.1 × 10-3 ppm) and 4.2 × 10-9 M (1.6 × 10-3 ppm), respectively, which were much lower than the maximum residue limits (MRLs) as reported in the European Union pesticides database as well as those from the U.S. Department Agriculture (MRLs are 0.01, 0.02 and 0.01 ppm for DDVP, malathion and ethion, respectively). These results suggested that this sensing system could be applied in the determination of different kinds of OPs. Table S2 listed different fluorescent probes for OPs detection, suggesting the sensitivity of this method was higher than that of 14
other fluorescent sensors. Furthermore, the reagents of this method were green and economical, for example, feather, H2O2, ammonia and CuSO4. 3.5. The anti-interference ability, stability and reproducibility To evaluate the selectivity of the proposed method, coexistence substances were added into the DDVP solution. The interference effect of 106-fold for NaCl and KCl, 800-fold for CaCl2 and Zn(Ac)2, 200-fold for FeCl3 and ascorbic acid, 104-fold for glucose, sodium acetate and sodium citrate, 200-fold for glycerol on the determination of 1.0 × 10 -8 M DDVP were studied (Fig. S6). The results showed that these coexistence substances had no influence on the signals of 1.0 × 10 -8 M DDVP with deviations below 5%. These results showed this method displayed good selectivity. To evaluate the stability of this sensor system, the prepared F-CDs was first kept in refrigerator at 4 ℃ for 30 days, then used in the determination of 1.0 × 10-8 M DDVP. The determination value was 98.8% of its initial response, which indicated that the method had good stability. To evaluate the reproducibility of this method, five parallel prepared F-CDs were using in the determination of 1.0 × 10-8 M DDVP. The relative standard deviation was 5.2%, which suggested that the sensor displayed good reproducibility. 3.6. Determination of DDVP in the cabbage and fruit juice samples In order to further investigate the accuracy, selectivity and repeatability of the proposed method, different concentrations of DDVP in the cabbage and fruit juice samples were determined (Table 1). From Table 1, it can be seen that the recoveries of 15
DDVP in these samples were in the range 94–104%, and the RSDs were less than 7.0%. The results revealed that this method was applicable in the determination of OPs in organophosphate compound-contaminated food samples.
