Food Science and Nutrition Studies ISSN 2573-1661 (Print) ISSN 2573-167X (Online) Vol. 3, No. 4, 2019 www.scholink.org/ojs/index.php/fsns 142 Original Paper A Synergistic Sensitized Fluorescent Determination of 2,4-Dichlorophenoxyacetic Acid in Vegetable Samples Based on the Derivatives of Calix[4]arene Xiashi Zhu1,2* & Qiuyi Ren2 1 College of Guangling, Yangzhou University, Yangzhou 225002, PR China 2 College of Chemistry & Chemical Engineering, Yangzhou University, Yangzhou 225002, PR China * Xiashi Zhu, College of Guangling, Yangzhou University; College of Chemistry & Chemical Engineering, Yangzhou University, Yangzhou 225002, PR China Received: October 24, 2019 Accepted: November 5, 2019 Online Published: November 18, 2019 doi:10.22158/fsns.v3n4p142 URL: http://dx.doi.org/10.22158/fsns.v3n4p142 Abstract A novel fluorescent determination of 2,4-dichlorophenoxyacetic acid (2,4-D) based on the derivatives of calix[4]arene (SAX) in β-cyclodextrin(β-CD)/ sodium dodecyl sulfate (SDS) synergistic sensitized system was developed. The results were shown that the fluorescence intensity of SAX could be quenched by 2,4-D, and the fluorescence quenching (∆F=FSAX-F2,4-D -SAX) was synergistic sensitized in β-CD/ SDS medium. Under the conditions of λex/em=332/468 nm and pH 7.0, the linear range for 2,4-D were found to be 0.020-4.00 μg/mL. The mechanism of determination was discussed with quenching type analysis, inclusion interaction and sensitizing effect. This method has been applied for the determination of 2,4-D in vegetable samples with satisfactory results. Keywords 2,4-dichlorophenoxyacetic acid, β-cyclodextrin, SDS, synergistic sensitization, fluorescence quenching 1. Introduction 2,4-dichlorophenoxyacetic acid (2,4-D, Figure 1(a)) belongs to the category of benzoic acid pesticides, which has the biological activity of auxin and can be used as plant growth regulator and preservative for vegetable and fruit (Jiang, Zha, & Tie, 2015). Nevertheless, 2,4-D residues in agricultural products and environment have great harm to human health due to its carcinogenic, mutagenic and estrogenic activity (Garabrant & Philbert, 2002). Up to now the reported techniques for 2,4-D determination have been performed on LC-MS (Jiang, Zha, & Tie, 2015), fluorescence spectroscopy (Wang, Yua, & Wu, 2016; www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 3, No. 4, 2019 143 Published by SCHOLINK INC. Boroduleva & Eremin, 2016; Atta, Bera, & Chattopadhyay, 2015), high performance liquid chromatography(HPLC) (Wu, Ee, & Lee, 2005), capillary electrophoresis (CE) (Zhu & Lee, 2001) and gas chromatography(GC) (Rezazadeh, Yamini, Seidi, Tahmasebi, & Rezaei, 2014). Although these techniques have good performance, they are complicated, expensive and time-consuming. So it is necessary to establish a rapid, simple and high selectivity method for 2,4-D detection. Calixarenes are macrocyclic compounds through a series of phenol connected with the ortho methylene units. In calixarene molecule, the upper edge is composed of para-position substituent of benzene; the lower edge is formed by neatly arranged phenolic hydroxyl group; the middle hydrophobic cavity is composed of benzene rings. Schiff base calix[4]arene (SAX, Figure 1(b)) has been followed with great interests due to the simple structure and high symmetry. The cavity of SAX is composed of four benzene rings, meeting the size and stability required for the inclusion interaction. The analytical method based on host-guest chemistry of calix[4]arene derivatives have been reported (Ma & Zhu, 2012; Yang, Yan, & Zhu, 2014; Wang, Zhu, & Yan, 2013; Yang, Qin, Yan, & Zhu, 2015; Li, X. Y., Li, M., & Chen, 2011; Khan, Shah, & Ahmed, 2016). But the fluorescence quenching methods using schiff base calix[4]arene derivatives as a fluorescent chemosensor for the determination of 2,4-D seems to be lacking. (a) (b) Figure 1. Chemical Structure of (a)2,4-D and (b)SAX The sensitivity of spectral analysis could be improved in suitable medium, such as surfactant (Ma & Zhu, 2012; Yang, Yan, & Zhu, 2014; Wang, Zhu, & Yan, 2013; Yang, Qin, Yan, & Zhu, 2015; Li, X. Y., Li, M., & Chen, 2011), β-cyclodextrin (β-CD) (Sanchez, Rubio, & Blanco, 1988; Márquez, Hernández, & García, 1990; Zhang, Liu, & Fan, 2009; Sánchez, Lopez, & Gómez, 1987; Zhu, Sun, Bao, & Guo, 2006; Sun, Zhu, & Wu, 2007) room temperature ionic liquid (Liu & Zhao, 2008; Berton & Martinis, 2009; Martinis & Olsina, 2008; Zhu & Jiang, 2011). In our previous publications, the sensitizing effects of surfactant (Wu, Ee, & Lee, 2005; Zhu & Lee, 2001; Rezazadeh, Yamini, Seidi, Tahmasebi, & Rezaei, 2014; Ma & Zhu, 2012), β-CD and its derivatives (Zhu, Sun, Bao, & Guo, 2006; Sun, Zhu, & Wu, 2007), ionic liquids (ILs) (Zhu & Jiang, 2011) on the ultraviolet spectrometry and spectrofluorimetry www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 3, No. 4, 2019 144 Published by SCHOLINK INC. were developed. Further research showed that mixed medium (such as surfactant/ILs, β-CD/surfactant) could synergistically sensitize fluorescence method, which has a better sensitization effect than that single medium (Zhu, Sun, Bao, & Guo, 2006; Zhu & Jiang, 2011; Ren & Zhu, 2016). In this study, the fluorescence intensity of SAX could be quenched by 2,4-D, the fluorescence quenching value (∆F=FSAX-F2,4-D-SAX) were enhanced in β-CD/SDS due to the synergistic sensitization, which has a much better quenching effect than that in single β-CD or SDS medium. There was a linear relationship between fluorescence quenching value (∆F) and concentration of 2,4-D, a novel β-CD/SDS synergistic sensitized fluorescence quenching method for the determination of 2,4-D was successfully developed. The mechanism of determination was also investigated. The proposed method was applied to analyte 2,4-D in real samples with satisfactory results. 2. Experimental Reagents and Instruments Schiff base calix[4]arene was synthesized according to the published methods (Bi, Sun, & Yan, 2012). 0.01% SAX (M=1328.7 g/mol, c = 7.5×10-6 mol/L) was prepared in ethanol. 100.0 µg/mL stock solution of 2,4-dichlorophenoxyacetic acid (2,4-D) was prepared by dissolving 0.100g 2,4-D in 100 mL volumetric flask and diluting with anhydrous ethanol to scale. The stock solutions were further diluted with anhydrous ethanol to obtain a standard working solution of 10.0 µg/mL for experiment. 1.0% β-CD solution was prepared by dissolving 1.00g of β-CD in 100.0 mL with distilled water. 1.0% SDS solution was prepared by dissolving 1.00g of SDS in 100.0 mL volumetric flask with distilled water. And pH=7.0 CH3COONH4 buffer solution was employed. All the fluorescence measurements were performed on a Hitachi F-4500 spectrofluorimeter (Japan) with excitation and emission slits at 10.0 nm and 5.0 nm, λex=332 nm. The pH was measured on a pH FE20 pH meter (Mettler Toledo). A UV 2501 spectrophotometer (Shimadzu, Japan) was used for all absorption spectral recordings and absorbance measurements . 3. Experiment Method Fluorescence measurements. In centrifuge tube (5.0 mL), 2.0 mL 0.01% SAX, 1.0 mL CH3COONH4 buffer solution (pH = 7.0), 0.5 mL 1.0% β-CD solution, 0.5 mL 1.0% SDS and 0.5 mL reference substance solution of 2,4-D (10.0 μg/mL) were added and then diluted to the scale with distilled water. Then fluorescence spectra was recorded in the range of 300- 650 nm with excitation at 332 nm. Quenching type analysis (Gong, Zhu, & Hu, 2007). 2.0 mL 0.01% SAX solution, 1.0 mL buffer solution, 0.5 mL 1.0% β-CD, 0.5 mL 1.0% SDS and different amount of 10.0 μg/mL 2,4-D solutions were added into 5.0 mL centrifuge tube, then diluted to the scale with distilled water and mixed completely. The fluorescence intensity of SAX was measured at different temperature (288 K, 298 K and313 K), respectively. Quenching type could be analyzed by Stern-Volmer Eq.(1): www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 3, No. 4, 2019 145 Published by SCHOLINK INC. QQ CKKC F F 0q 0 11  F0 and F were the fluorescence intensities of SAX in the absence and presence of 2,4-D respectively, K was the Stern–Volmer quenching constant, CQ was the concentration of quencher 2,4-D, Kq was the quenching rate constant, τ0 was the average lifetime of the SAX without 2,4-D. If the quenching type is single static or dynamic quenching, the curve of F0/F versus CQ (Stern–Volmer curve) would be linear within certain concentration. Absorption spectrum titrations (Fu, Zeng, & Mu, 2012). The recognition ability of SAX and β-CD to 2,4-D can be evaluated through the change of absorption spectrum. The absorption spectrum titrations of 2,4-D with SAX and β-CD was made in the range of 200.0-600.0 nm. The absorbance of 2,4-D was measured with nSAX: n2,4-D and nβ-CD: n2,4-D. Inclusion interaction. The solution of a certain amount of SAX, 1.0 mL buffer solution and different amount of 10.0 μg/mL 2,4-D solutions were added into 5.0 mL centrifuge tube, then diluted to the mark with distilled water and mixed thoroughly. The fluorescence intensity was measured at 25℃, then the Benes-Hildebrand method (Vimal, Ajay, & Narinder, 2008) (double reciprocal plot) was used to calculate the inclusion constant (K) of SAX-2,4-D and β-CD-2,4-D assuming a 1:1 inclusion model. The Benesi-Hildebrand method is a spectroscopic method to determine the inclusion constants of the host guest complexes (including fluorescence spectroscopy and absorption spectroscopy). The equation is as follows (SAX as an example):        00 14,2111 SAXDSAXKF   where [SAX]0 was the concentration of SAX, ∆F was the quenching value of fluorescence intensity, α was a constant. Thus, the inclusion constant (K) of the 1:1 inclusion complex could been calculated by dividing the intercept by the slope of the double reciprocal plot. Determination of critical micelle concentration (cmc). cmc values of the medium were measured by conductivity measurements. The cmc was obtained from the inflection point of the straight lines of before and after micellar concentration range (Kumaraguru & Santhakumar, 2006; Mehta, Bhawna, & Ram, 2010). Determination of fluorescence quantum yield. Fluorescence quantum yields of SAX were measured using 1.0×10−6 g/mL quinine sulfate as reference substance (Zhao & Wei, 2006; Zhu, Gong, & Yu, 2008). Under the same apparatus conditions, the quantum yield of the SAX was calculated. Kinetics of the reaction. The kinetics of the reaction could be described by the first order kinetic model, the second kinetic model and the Weber Maurice diffusion model (Azizian & Fallah, 2010; Wang, Wei, & Li, 2015). In this study, the first order kinetic fitting of the quenching process was carried out. Sample preparation. Vegetable samples (green vegetables, Chinese cabbage and chrysanthemum coronarium) were purchased from local market. A certain amount of each vegetable sample was cut up www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 3, No. 4, 2019 146 Published by SCHOLINK INC. and homogenized. Then, 20 g of each sample was weighed and placed in a 50 mL centrifuge tube and 50 mL ethanol was added as well. Then tighten up the lid, dipped for 1 hour, shaken thoroughly for 20 min. After centrifugation 10 min (5000 r/min), the test sample solution was prepared after the upper solution was filtered. In order to reduce the fluorescence background, the sample solution was diluted 15 times and then used as an analytical sample (Farokhcheh & Alizadeh, 2013). 4. Results and Discussion Choice of medium. The effect of different medium on ∆F (∆F = FSAX− F2,4-D–SAX) was studied. As can be seen in Figure 2 that the sequence of ∆F was ∆Fβ-CD-SDS>∆FSDS>∆Fβ-CD > ∆FH2O. The fluorescence quenching value in β-CD/ SDS synergistic sensitized medium was greater than that in single β-CD or SDS medium. So β-CD-SDS medium was selected for further experiment. 1 2 3 4 0 100 200 300 400 500 F H 2 O SDS -CD -CD-SDS Figure 2. Effect of Different Medium on Fluorescence Intensity Fluorescence spectra. The fluorescence emission spectrum of SAX (present or absent of 2,4-D) in β-CD-SDS and H2O medium were shown in Figure 3. It can be seen that (1) the fluorescence intensity of SAX (FSAX) was enhanced in β-CD/SDS medium (curves a and c); (2) the fluorescence intensity of SAX (FSAX or FSAX-β-CD-SDS) was quenched when 2,4-D was added (curves b and d) and gradually diminished with the increase concentration of 2,4-D (inset Figure 3); (3) ∆F= FSAX-β-CD-SDS – F2,4-D-SAX-β-CD-SDS was larger than that ∆F’=FSAX-F2,4-D-SAX with the same concentration of 2,4-D, which was the synergistic sensitizing effect in β-CD/SDS. www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 3, No. 4, 2019 147 Published by SCHOLINK INC. 350 400 450 500 550 600 650 0 500 1000 1500 2000 2500 3000 350 400 450 500 550 600 650 0 500 1000 1500 2000 2500 3000 F nm 1 8 F ' nm F a d F Figure 3. Fluorescence Spectra (a). SAX-β-CD-SDS (b). 2,4-D-SAX-β-CD-SDS (c). SAX-H2O (d). 2,4-D-SAX-H2O Inset 1-8: 2,4-D-SAX-β-CD-SDS, [2,4-D]: (1) 0 µg/mL, (2) 0.02µg/mL, (3) 0.08µg/mL, (4) 0.1µg/mL, (5)0.2µg/mL, (6) 0.4µg/mL, (7) 0.8µg/mL, (8) 1.0 µg/mL Effect of pH. The influence of pH on ∆F was investigated. As could be seen in Figure 4, ∆F gradually increased with the increase of pH and reached maximum at pH = 7.0, but it diminished at pH > 7.0. The reason may be related to the formation of SAX-2,4-D inclusion, which will be discussed in section 3.13.2. So 1.0 mL of pH = 7.0 CH3COONH4 buffer solution was chosen for the further study. 4 5 6 7 8 9 10 0 20 40 60 80 100 pH F(a.u) Figure 4. Effect of pH on Fluorescence Quenching Value Effect of SAX amount. The effect of the amount of SAX was studied in Fig. 5. It was shown that the ∆F was increased and reached a maximum value at a SAX (7.5×10-6 mol/L) amount of 2.0 mL, and then decreased (curve 1). This was because that the FSAX-β-CD-SDS gradually decreased with the increase of SAX amount due to the self-quenching of SAX at higher concentration (curve 2). Thus, 2.0 mL SAX (7.5×10-6 mol/L) was selected for the optimized method. www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 3, No. 4, 2019 148 Published by SCHOLINK INC. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 20 40 60 80 100 120 140 160 180 200 SAX/mL F(a.u) 0 500 1000 1500 2000 2500 3000 F 2 1 Figure 5. Effect of the Amount of SAX on Fluorescence Quenching Value (Left Axis) and Fluorescence Intensity of SAX (Right Axis) (1: fluorescence quenching value, 2: fluorescence intensity of SAX) Effect of β-CD amount. Effect of β-CD was investigated. As is shown in Fig. 6, with the increase of β-CD (1.0%) amount, ∆F gradually increased and reached the maximum when the β-CD amount was 0.50 mL, but decreased when β-CD amount was more than 0.50 mL. The change trend of ∆F could be explained from the change of FSAX-β-CD-SDS and F2,4-D-SAX-β-CD-SDS with β-CD amount (inset Figure 6). (1) FSAX-β-CD-SDS gradually increased with β-CD amount while F2,4-D-SAX-β-CD-SDS slowly decreased (Vβ-CD = 0-0.50 mL) ( ∆F↑= FSAX-β-CD-SDS↑ -F2,4-D-SAX-β-CD-SDS↓); (2) FSAX-β-CD-SDS gradually decreased, while FCR-SAX-M-β-CD-Tx-100 remain unchanged ( Vβ-CD = 0.50-0.80 mL) (∆F↓= FSAX-β-CD-SDS↓ -F2,4-D-SAX-β-CD-SDS). Therefore, 0.50 mL 1.0% β-CD was chosen for the following experiments. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0 20 40 60 80 100 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 500 1000 1500 2000 2500 3000 3500 2,4-D-SAX--CD-SDS SAX--CD-SDS -CD/mL F F(a.u) -CD/mL Figure 6. Effect of the Amount of β-CD on Fluorescence Quenching Value Effect of SDS amount. Effect of SDS was studied. As is shown in Figure 7, ∆F gradually rose up with the increase of SDS (1.0 %) amount and up to the maximum when the SDS amount was 0.50 mL, but ∆F decreased when SDS amount was greater than 0.50 mL. www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 3, No. 4, 2019 149 Published by SCHOLINK INC. The change of FSAX-β-CD-SDS and F2,4-D-SAX-β-CD-SDS with SDS amount (inset Figure 7) could declare the change of ∆F with SDS. (1) FSAX-β-CD-SDS obviously increased with SDS amount was larger than F2,4-D-SAX-β-CD-SDS ( VSDS = 0-0.30 mL)( ∆F↑= FSAX-β-CD-SDS↑↑ - F2,4-D-SAX-β-CD-SDS↑); (2) FSAX-β-CD-SDS continuously increased, while F2,4-D-SAX-β-CD-SDS gradually decreased ( VSDS= 0.30–0.50 mL) (∆F↑= FSAX-β-CD-SDS↑-F2,4-D-SAX-β-CD-SDS↓); (3) FSAX-β-CD-SDS gradually decreased, while F2,4-D-SAX-β-CD-SDS changed rarely (VSDS= 0.50–0.80 mL) (∆F↓= FSAX-β-CD-SDS↓ -F2,4-D-SAX-β-CD-SDS). Therefore, 0.50 mL 1.0 % SDS was chosen for the further study. In conclusion, the ∆F was biggest when mass ratio or molar ratio of β-CD and SDS are 1:1 and 1:4, respectively, which illustrated that β-CD and SDS showed the synergistic sensitized effect on ∆F. Either excess β-CD or SDS will weaken the synergistic sensitized effect, resulting the decrease of ∆F. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0 20 40 60 80 100 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 500 1000 1500 2000 2500 3000 3500 2,4-D-SAX--CD-SDS SAX--CD-SDS SDS/mL F SDS/mL F(a.u) Figure 7. Effect of the Amount of SDS on Fluorescence Quenching Value Effect of reaction time. The effect of reaction time on ∆F was tested. It was found from Figure 8 that ∆F obviously increased from 10 min to 30 min, then ∆F remained unchanged after 30 min. Therefore, 30 min of reaction time was chosen for the following experiments. According to the data ∆F-t, the fluorescence quenching reaction kinetics could be discussed. In this study, the first order kinetic fitting of the quenching process was carried out. As shown in inset Fig.8, the curve of lnC2,4-D versus t was linear and R2 was 0.9960. The reaction rate constant was 1.14×10-1 min-1. The results show that the fluorescence quenching process followed the first order kinetic model. The reason may be that the amount of SAX is excess in this system, so the rate of fluorescence quenching reaction is only in relation to the concentration of 2,4-D. www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 3, No. 4, 2019 150 Published by SCHOLINK INC. 0 20 40 60 80 100 0 20 40 60 80 100 5 10 15 20 25 -3.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 t/min ln(CA) F(a.u) t/min First order kinetics model Figure 8. Effect of Reaction Time on Fluorescence Quenching Value Effect of temperature. The effect of temperature (5-50℃) on ∆F was investigated. As can been seen from Figure 9 that ∆F was increased from 5 to 25℃ then it decreased with the increase of temperature. Thus, the suitable temperature of 25℃ was chosen for the study. 0 10 20 30 40 50 0 20 40 60 80 100 T/℃ F(a.u) Figure 9. Effect of Temperature on Fluorescence Quenching Value Effect of foreign substances. The effect of foreign substrates was discussed for the determination of 1.0 μg/mL 2,4-D. With a relative error of less than ±5%, the majority of these substances showed no remarkable interference in the determination of 2,4-D (Table 1). Table 1. Effect of Interfering Substances Foreign substances Foreign substances / 2,4-D(w/w) Foreign substances Foreign substances / 2,4-D(w/w) K+ 500 Al3+ 20 Na+ 1200 Mn2+ 2 Ca2+ 1000 Cl- 1800 www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 3, No. 4, 2019 151 Published by SCHOLINK INC. Mg2+ 300 NO3 - 700 Ba2+ 100 SO4 2- 300 Zn2+ 200 Glucose 500 Fe3+ 2 Sucrose 500 Cu2+ 2 Glycine 200 Analytical performance. Under the optimum conditions, the linear regression equations were determined to be: ΔF = 10.14 + 2672.1c (μg/mL) in the range of 0.020~0.10 μg/mL, R = 0.9972, the detection limit estimated (S/N = 3) was 0.23 ng/mL; ΔF = 331.08 + 290.87c (μg/mL) in the range of 0.10μg/mL ~ 4.0 μg/mL, R = 0.9918, the detection limit estimated was 2.8 ng/mL. The relative standard deviation (RSD) was 0.96% (n = 3, c = 1.0μg/mL). Sample analysis. The proposed method was successfully applied for the determination the amount of 2,4-D in vegetable samples. The data were listed in Table 2. The recovery ratio ranged from 95.0% ~ 105.5% which was satisfactory. Table 2. Determination Results of 2,4-D Samples Added (μg/g) Found（μg/g） Recovery (%) Chinese cabbage 0.0 ND - 0.50 0.53 105.5 1.0 0.95 95.5 2.0 1.9 98.2 green vegetables 0.0 ND - 0.50 0.48 95.0 1.0 2.0 95.7 2.0 2.1 102.0 chrysanthemum coronarium 0.0 ND - 0.50 0.52 104.9 1.0 0.96 96.5 2.0 2.1 103.4 Comparison of different methods. The results obtained from this experiment were compared with those previously reported methods for 2,4-D determination (Table 3). The advantages of the proposed method are: easy operation, high sensitivity, low detection limit and high recovery rate. www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 3, No. 4, 2019 152 Published by SCHOLINK INC. Table 3. Comparison with Previously Reported Methods Methods Linearity range LOD Recovery Reference HPLC-MS 0.025-1.0 mg/L 0.005 mg/kg 88.3-95.4% (Jiang, Zha, & Tie, 2015) SHPLC 1.0-500 ng/mL 0.3 ng/mL 105-116 % (Wu, Ee, & Lee, 2005) CE 3.0-500 ng/mL 0.02 ng/mL - (Zhu & Lee, 2001) GC 10-500 ng/mL 5.0 ng/mL - (Rezazadeh, Yamini, Seidi, Tahmasebi, & Rezaei, 2014) Fluorescence quenching calix[4]arene methods 0.020-4.0 µg/mL 2.8 ng/mL 95.0-105.5% This method 5. Discussion of Mechanism In this paper, the discussion of mechanism was included quenching type analysis, inclusion interaction and sensitizing effect. Quenching type. Quenching types can be divided into static quenching and dynamic quenching. The static quenching is caused by the formation of non or weak fluorescent compound (Gong, Zhu, & Hu, 2007). The dynamic quenching is initiated from the collision of fluorescence substance and quencher, resulting in the decrease of fluorescence intensity and quantum yield. Quenching type could be discussed with Ksv and Kq. With the increasing temperature, Ksv would be decreased for static quenching, while Ksv would be increased for dynamic quenching (Gong, Zhu, & Hu, 2007). The Stern-Volmer plots of SAX with 2,4-D at different temperature (288 K, 298 K and 313 K) was shown in Figure 10. The order of Ksv at different temperature were Ksv 288K =3.90×104 > Ksv 298K =3.45×104 > Ksv 313K=3.04×104 L/mol. It demonstrated that the fluorescence quenching mechanism of SAX by 2,4-D was a static quenching procedure and a complex was formed between SAX and 2,4-D. 0.0 0.5 1.0 1.5 2.0 0.95 1.00 1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 C 2,4-D (g/mL) F0/F a b c Figure 10. The Stern-Volmer Curves for the Binding of 2,4-D with SAX at 288 K (a), 298 K (b) and 313 K (c) www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 3, No. 4, 2019 153 Published by SCHOLINK INC. Inclusion interaction. From the previous discussion, the type of fluorescence quenching was static quenching, which may be caused by the formation of non or weak fluorescent compound. SAX and β-CD have a cavity structure, both of them are likely to form inclusion interaction with 2,4-D. The recognition ability of SAX and β-CD to 2,4-D can be evaluated through the change of absorption spectrum. The results of an absorption titration at λ = 281.0 nm (characteristic absorption peak of 2,4-D) (Figure 11) was shown that the absorbance of 2,4-D was gradually increased with the increase of SAX or β-CD and was unchanged when nSAX:n2,4-D = 1:1 or nβ-CD:n2,4-D = 1:1, which implied a 1:1 stoichiometry for binding between SAX and 2,4-D or β-CD and 2,4-D. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 0.04 0.05 0.06 0.07 0.08 0.09 0.10 0.11 0.12 n/n2,4-D A 1 2 Figure 11. Absorption Spectrum Titrations of 2,4-D with SAX and β-CD (1: 2,4-D-SAX, 2: 2,4-D-β-CD) The inclusion constant (K) of SAX-2,4-D and β-CD-2,4-D could be obtained by Benesi-Hildebrand method (Vimal, Ajay, & Narinder, 2008). The larger the value of K, the more steady the inclusion complex. As is shown in Figure 12, double reciprocal plots of SAX-2,4-D and β-CD-2,4-D have good linear relationship, which supports the formation of a 1:1 complex. The calculated inclusion constant (K) was listed in Table 4. As can be seen in Table 4: (1)K SAX-2,4-D >>Kβ-CD-2,4-D, which suggested that inclusion complex of SAX-2,4-D is more stable than β-CD-2,4-D. SAX plays a major role in inclusion interaction with 2,4-D, which bringing about static fluorescence quenching. (2) K SAX-2,4-D > K SAX-2,4-D-β-CD , the inclusion constant of SAX-2,4-D was decreased in presence of β-CD, which is caused by the formation of β-CD-2,4-D complex; (3) K SAX-2,4-D, pH 7.0 > K SAX-2,4-D, pH 4.5 > K SAX-2,4-D, pH 10.0, which indicated that pH had a significant effect on the formation SAX-2,4-D and the inclusion complex of SAX-2,4-D is more stable at pH 7.0 than in acidic or basic solution; (4) the larger the K, the greater the ∆F, the largest fluorescence quenching value ∆F was at pH=7.0, which is in accordance with the discussion about effect of pH on ∆F in section 3.3. www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 3, No. 4, 2019 154 Published by SCHOLINK INC. 0 200000 400000 600000 800000 1000000 1200000 0.000 0.002 0.004 0.006 0.008 0.010 0.012 1/c 1/F a 0 200000 400000 600000 800000 1000000 1200000 0.000 0.002 0.004 0.006 0.008 0.010 0.012 0.014 0.016 0.018 1/F 1/c b 0 1000 2000 3000 4000 5000 0 200 400 600 800 1000 1/C A c Figure 12. Benesi-Hildebrand Plot (a: SAX-2,4-D, b: SAX-2,4-D-β-CD, c: β-CD-2,4-D) Table 4. Results of Inclusion Constant System Inclusion constant K（L/mol） pH 4.5 pH 7.0 pH 10.0 SAX-2,4-D 5.71×104 5.55×1010 2.00×104 SAX-2,4-D-β-CD 2.21×109 β-CD-2,4-D 262.47 Sensitizing effect. The discussion of sensitizing effect was included the formation of the ordered molecular assembly and fluorescence quantum yield. The formation of the ordered molecular assembly The synergistic sensitized effect of β-CD/SDS may be from the formation of the ordered molecular assembly. The interaction of SDS and β-CD can change the critical micelle concentration (cmc). By measuring the cmc of a single and mixed medium, it can be determined whether the β-CD/SDS has formed a new ordered molecular assembly (Zhu & Jiang, 2011). The value of cmc can be obtained from the inflection point of the straight lines of before and after micellar concentration range (Kumaraguru & www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 3, No. 4, 2019 155 Published by SCHOLINK INC. Santhakumar, 2006). The results were summarized in Figure 13 and Table 5. As is shown in Figure 13, the cmc of β-CD and SDS/β-CD were 3.0-5.0 mmoL/L and 5.0-6.0 mmoL/L, respectively. The cmc of the mixed medium model could be calculate by the clint model based on the assumption of ideal mixture behavior. As is shown in Table 5, the cmc of SDS is 8.4 mmoL/L (Mehta, Bhawna, & Ram, 2010), the molar ratio of SDS and β-CD was 4:1. Thus, the cmc of the mixed solution should between 7.8-8.1 mmoL/L. There was a difference between the cmc of the mixed medium (β-CD/SDS) in determined value and the clint model calculated value. This result illustrated that the β-CD/SDS mixed medium formed a new ordered molecular assembly. What’s more, the sensitivity of the determination could be enhanced by the new ordered molecular assembly. 0 2 4 6 8 10 12 14 16 18 0 200 400 600 800 1000 concentration mmoL.L-1 th e el ec tr ic c o n d u ci ti vi ty (  s. cm -1 ) a b Figure 13. Electrical Conductivity vs. the Concentration of β-CD (a: Electrical conductivity vs. the concentration of β-CD, b: Electrical conductivity vs. the concentration of SDS/β-CD) Table 5. cmc of the Different Medium Medium cmc (mmoL/L) Determination value Clint model value SDS 8.4a β-CD 3.0-5.0 SDS/β-CD (4:1) 5.0-6.0 7.8-8.1b a: the literature (Mehta, Bhawna, & Ram, 2010); b: calculate by Clint model Fluorescence quantum yield. The fluorescence quantum yield represents the ability of translation of absorption energy to fluorescence. It is one of the basic and significant parameters for fluorescence substances. What’s more, ∆F = FSAX – F2,4-D-SAX, where ∆F must rise up with the increase of fluorescence quantum yield of SAX. The fluorescence quantum yield of SAX (Y) in the medium of www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 3, No. 4, 2019 156 Published by SCHOLINK INC. H2O，β-CD，SDS and β-CD-SDS were listed in Table 6. As can be seen in Table 6, the order of fluorescence quantum yield of SAX in different medium is Yβ-CD-SDS > YSDS > Yβ-CD > YH2O. It is in accord with the sensitivities in different medium. The results could be considered with internal and external environment. The fluorescence quantum yield of SAX in β-CD-SDS medium was increased obviously. It was because that the external environment provided by β-CD/SDS mixed medium is more favorable on solubilization than that in single medium (β-CD or SDS). Moreover, the microenvironment which was formed by the new ordered molecular assembly β-CD/SDS could provide the protective environment for the singlet excited state and could reduce the non-radiation of the fluorescent substance SAX. In other words, both self fluorescence quenching of SAX and fluorescence quenching of external quenchers can be decreased in the medium of β-CD/SDS. As a result, the fluorescence quantum yield of SAX in the mixed medium is largest due to the synergistic sensitizing effect of β-CD-SDS. Table 6. The Fluorescence Quantum Yield of 2,4-D Medium Y H2O 0.011 β-CD 0.038 SDS 0.094 β-CD-SDS 0.108 In summary, the mechanism of this method was (1) the fluorescence quenching of SAX is due to the inclusion interaction of SAX-2,4-D; (2)the sensitizing effect is that SDS/β-CD medium could form a new ordered molecular assembly, in which fluorescence quantum yield of SAX increased obviously, resulting synergistic sensitizing effect on fluorescence quenching value. 6. Conclusion In this study, a novel fluorescence quenching method for the determination of 2,4-D has been developed. The fluorescence intensity of SAX was quenched due to inclusion interaction between SAX and 2,4-D, and the fluorescence quenching value (∆F) was increased in β-CD-SDS medium. The proposed method has been applied for the determination of 2,4-D in vegetable samples with satisfactory results. Funding The National Natural Science Foundation of China (21375117) and the Priority Academic Program Development of Jiangsu Higher Education Institutions are acknowledged for funding this research. www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 3, No. 4, 2019 157 Published by SCHOLINK INC. References Atta, S., Bera, M., & Chattopadhyay, T. (2015). Nano-pesticide formulation based on fluorescent organic photoresponsive nanoparticles: For controlled release of 2,4-D and real time monitoring of morphological changes induced by 2,4-D in plant systems. RSC Adv., 5, 86990-86995. https://doi.org/10.1039/C5RA17121K Azizian, S., & Fallah, R. N. (2010). 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