A highly sensitive octopus-like azobenzene fluorescent probe for determination of abamectin B1 in apples

The development of detecting residual level of abamectin B1 in apples is of great importance to public health. Herein, we synthesized a octopus-like azobenzene fluorescent probe 1,3,5-tris (5′-[(E)-(p-phenoxyazo) diazenyl)] benzene-1,3-dicarboxylic acid) benzene (TPB) for preliminary detection of abamectin B1 in apples. The TPB molecule has been characterized by ultraviolet–visible absorption spectrometry, 1H-nuclear magnetic resonance, fourier-transform infrared (FT-IR), electrospray ionization mass spectroscopy (ESI-MS) and fluorescent spectra. A proper determination condition was optimized, with limit of detection and limit of quantification of 1.3 µg L−1 and 4.4 μg L−1, respectively. The mechanism of this probe to identify abamectin B1 was illustrated in terms of undergoing aromatic nucleophilic substitution, by comparing fluorescence changes, FT-IR and ESI-MS. Furthermore, a facile quantitative detection of the residual abamectin B1 in apples was achieved. Good reproducibility was present based on relative standard deviation of 2.2%. Six carboxyl recognition sites, three azo groups and unique fluorescence signal towards abamectin B1 of this fluorescent probe demonstrated reasonable sensitivity, specificity and selectivity. The results indicate that the octopus-like azobenzene fluorescent probe can be expected to be reliable for evaluating abamectin B1 in agricultural foods.

Avermectins, being one type of macrolide antibiotics, have been widely used as bactericide, insecticide and miticide for plants or animals, which have excellent characteristics of disturbing the target's neurophysiological activities and can easily be decomposed by soil microorganisms 1 . Abamectin B 1 , is the only avermectin that has been widely approved for plants and animals because of its efficient antiparasitic activity 2,3 . However, the spread use of avermectins tends to result in consecutive accumulation in food-producing animals and plants 4,5 . To detect avermectins (Abamectin B 1 ), various analytical approaches were employed, involving high-performance liquid chromatography-ultraviolet detection (HPLC-UV) 4 , liquid chromatography-tandem mass spectrometry (LC-MS) 6 , high-performance liquid chromatography-fluorescent detector (HPLC-FLD) 7 , enzyme-linked immunosorbent assay (ELISA) 8 and liquid chromatography-tandem mass spectrometry/mass spectrometry (HPLC-MS/MS) 9 . Whereas, in most case, these approaches either needed a time-consuming process, high-cost accurate instrument, and high professional operators, which makes them difficult to apply in general laboratories 10 . Herein, considering the potential harm of abamectin B 1 for public health, a convenient and accurate analysis method for residual abamectin B 1 is necessary 10,11 . Fluorescent probe has gained great attention in recent years owing to high sensitivity, high selectivity, fast response, low cost, and direct detection 12 . Especially, azobenzene fluorescent probe exhibits outstanding fluorescent quantum yield, light stability 13 , and chemical and thermal stability 14 . Fluorescent probe has been applied to analyze organophosphorus pesticides 15 , organochlorine pesticides 16 and carbamate pesticides 17 . However, there are few reports on fluorescent probe for monitoring avermectin residual in literature due to the weaker fluorescence signal of a single chromophore 18 and the difficulty in recognizing their complicated chemical structures containing ketones, aldehydes and hydroxyl groups 19 . Therefore, a fluorescent molecule, with rationally designed structures, containing multiple chromophores and recognition groups, is an ideal probe to monitor avermectin B 1 . www.nature.com/scientificreports/ Herein, this study reported the synthesis of octopus-like 1,3,5-tris (5′-[(E)-(p-phenoxyazo) diazenyl)] benzene-1,3-dicarboxylic acid) benzene (TPB), with six carboxyl groups and three azo chromophores (Fig. 1). Its application as a fluorescent probe was proved to be feasible by evaluating its fluorescence properties. A sensitive and specific approach was further established for qualitative and quantitative assay of abamectin B 1 . The applicability of approach was evaluated in apple samples, based on a visible fluorescence signal for this probe towards avermectin B 1 at 420 nm.

Results and discussion
Fluorescence properties of TPB. The fluorescence property of TPB was investigated, with its precursor 5-(4-hydroxyphenylazo)-isophthalic acid dimethyl ester (DDH) (chemical structure was shown in Figure S1) for comparison, which only has one azo chromophore. When TPB and DDH were irradiated by ultraviolet light, TPB displayed a maximum emission at 350 nm and excitation at 290 nm (Fig. 2), while no fluorescence signal was visible for DDH (Fig. 3). The difference of fluorescence property was attributed to the more released energy of TPB than that of DDH when the excited state electrons returned from the excited singlet state (S) to the spin singlet electron (S 0 ), which was based on the superposition of fluorescence effect of three azo chromophores. Furthermore, improved fluorescence property of TPB was ascribed to enhance resistance to internal rotation of molecule resulting from the introduction of 1,3,5-tris (bromomethyl) benzene and the expansion of space system effect. Moreover, the Stokes shift of TPB was calculated to be 60 nm. The Stokes shift, was considered to  www.nature.com/scientificreports/ effectively decrease detection errors, resulting from the interference from auto-fluorescent of samples 24 and the spectral overlap between the fluorescent and excitation light 25 . Therefore, TPB was expected to be suitable for employ as a fluorescent probe. We further investigated the applicability of TPB to abamectin B 1 . Upon the addition of abamectin B 1 , the emission band at 350 nm and the excitation band at 290 nm of the probe shifted forward to 420 nm and 360 nm respectively (Fig. 3). Generally, different fluorescence molecules have different excitation and emission spectra, which can determine the specificity and selectivity of analysis by using fluorescent probe. The Stokes shift of 60 nm was calculated and reflected rational anti-interference ability. This result confirmed that abamectin B 1 could be identified by probe TPB under the certain conditions, which indeed underpinned qualitative analysis of abamectin B 1 .
Furthermore, the fluorescence intensities of the reacted product at 420 nm were examined at different concentrations of abamectin B 1 . Figure 4 presented that the fluorescence intensity of the reacted product at 420 nm were enhanced gradually with the increasing concentration of abamectin B 1 . The which was considered as the basis of quantitative analysis. Therefore, TPB could be used as a fluorescence probe for assessing the level of abamectin B 1 .   Fig. 4), which confirmed the probe TPB was the typical fluorescent molecule based on the effect of Photoinduced Electron Transfer (PET). Further analysis for TPB' structure showed that its carboxyl groups, azo groups and peripheral phenyls might be considered as receptors, fluorophores and spaces respectively. Based on frontier orbital theory, in the existence of abamectin B 1 and its relevant substances with the same recognized group, probe TPB should engender different LUMO and HUMO, which leaded to unequal excitation and emission spectra. Therefore, probe TPB has a good sensing selectivity.
The mechanism of identifying abamectin B 1 by TPB. To investigate the mechanism of identifying abamectin B 1 by TPB, fluorescence spectroscopy, FT-IR and ESI-MS were employed to compare the difference of the TPB before and after adding abamectin B 1 . After adding abamectin B 1 , FT-IR spectra showed additional peaks at 1704 cm −1 and at 1149 cm −1 ( Figure S2), corresponding to the ester group. The appearance verified the recognition of probe TPB to abamectin B 1 achieved by esterification. In addition, a clear red shift of wavelengths in fluorescence spectroscopy was noticed (Fig. 3), which was assigned to the changed conjugate systems of electron-donating and rearranged internal charges prompted by enhanced ability of TPB to capture abamectin B 1 . Moreover, ESI-MS spectra yielded a peak at m/z = 2717.1910 ( Figure S6), which corresponded to a new product produced between probe TPB and abamectin B 1 , thus underlying the mechanism of detection.
For abamectin B 1 , the hydroxyl groups at the C 5 and C 7 are allylic, which should have the high reactive. However, the allylic hydroxyl group at the C 7 A is too easily forming hydrogen bonds with adjacent ester groups and too sterically hindered to be reactive 26 . Hydroxyl group at the C 4 is a general, and its activity is weaker than that of C 5 and C 7 . Thus, only hydroxyl group at the C 5 has the potential to be used for esterification theoretically. In addition, due to the effect of steric hindrance on TPB, its combination with abamectin B 1 can only take place in the counterpoint. However, on the same phenyl of TPB with two carboxyl groups, in the existence of one abamectin B 1 , the adjacent carboxyl groups will be passivated and incapable to add another abamectin B 1 . Therefore, a recognition mode was tentatively proposed, i.e., the two carboxyl groups of the octopus-like azobenzene fluorescent probe TPB were used as recognition sites to abamectin B 1 with a molar ratio of 1:2 (Fig. 5), which was further confirmed by ESI-MS spectra with a peak at m/z = 2717.1910 ( Figure S6).

Establishment and evaluation of the method.
To optimize the determination conditions, effects of different levels of the pH and amount of phosphate buffer were investigated. Figure S7 showed that the fluorescent intensity of the reacted product at 420 nm was initially increased at pH 5.0-6.0, followed by a decrease at pH 6.0-9.0, and eventually by the maximum at pH 6.0. The increase-decrease-maximum trend reflected the proper protonation of carboxyl groups of TPB facilitated the coordination process and enhanced the nucleophilicity of hydroxyl of TPB to abamectin B 1 . Figure S8 showed that the fluorescent intensity of the product at 420 nm was initially increased at 0.2-0.8 mL phosphate buffer (0.2 M, pH 6.0), followed by a stabilization at 0.8-1.2 mL phosphate buffer (0.2 M, pH 6.0). Therefore, the validation of using probe TPB was estimated at 0.8 mL phosphate buffer (0.2 M, pH 6.0), by optimizing the determination conditions, linear equation, correlation coefficients (R 2 ), limit of detection (LOD), limit of quantification (LOQ) precision and linear range. Apparently, the fluorescent  (Table S2). In addition, the LOD of the described method was calculated as 1.3 µg L −1 (Table S2). The linear regression equation was thus determined to be Y = 3987X -0.7143 (Fig. 6), where Y was the fluorescent intensity of probe TPB at 420 nm, and X represented the concentration of abamectin B 1 . Correlation coefficients (R 2 ) was determined to be 0.9997, which manifested the satisfactory precision of the method. The enhancement clarified the suitability of this convenient and sensitive method for the determination of the abamectin B 1 with tiny LOD, miniature LOQ, excellent precision and epic linear range.
Further comparison was conducted between our fluorescent probe method and those described previously. Table S3 showed that the recovery, linear range and LOQ of our fluorescent probe method were comparable to or superior to those of the methods reported previously.
A good reproducibility was represent by a relative standard deviation of 2.2% between eleven parallel experiments shown in Table S2. The final product had an unique fluorescence spectra at EX of 360 nm and EM of 420 nm, which could be considered to embody the specificity and selectivity of the method generally, as mentioned above. Sensitivity of the method was benefit from the six carboxyl recognition sites and three azo response sites of TPB. Table 1 (Table 1), which indicated the suitability of this detection method in apples.

Conclusion
In summary, we had demonstrated the probe TPB synthesized for facile quantitative detection of the residual abamectin B 1 in apples. The octopus-like TPB molecule was characterized using UV-Vis, 1 H NMR, FT-IR, ESI-MS and fluorescent spectra. The determination conditions were tuned by varying different pH value and concentration, and a proper condition was achieved at pH 6.0 with LOD and LOQ of 1.3 µg L −1 and 4.4 μg L −1 , respectively. The mechanism of the probe to identify abamectin B 1 was tentatively proposed in a aromatic nucleophilic substitution through a combined mode of TPB and abamectin B 1 with a molar ratio of 1:2. In particular, the facile quantitative detection of the residual abamectin B 1 in apples was achieved. Our results showed that the novel approach of quantitative assay based on fluorescent probe is significative to evaluate the abamectin B 1 in agricultural foods.

Materials and methods
Reagents and materials. Dimethyl   Preparation of probe TPB. TPB was synthesized by the scheme showed in Figure S1 based on a previously reported method 20 , which was confirmed by FT-IR, UV-Vis, 1 Table S1.
General procedure for fluorescent spectra measurement. Tetrahydrofuran, as a solvent, was used to prepare the probe TPB solution (0.06 mmol L −1 ), abamectin B 1 solution (1.00 mg L −1 ), and DDH solution (0.06 mmol L −1 ). 0.5832 g TPB was added to a volumetric flask and mixed with tetrahydrofuran solution to 100 mL to get probe TPB solution (6.00 mmol L −1 ). Then, 1 mL TPB (6.00 mmol L −1 ) was added to a volumetric flask and mixed with tetrahydrofuran solution to 100 mL to get probe TPB solution (0.06 mmol L −1 ). 0.1716 g DDH was added to a volumetric flask and mixed with tetrahydrofuran solution to 100 mL to get probe DDH solution (6.00 mmol L −1 ). Then, 1 mL DDH (6.00 mmol L −1 ) was added to a volumetric flask and mixed with tetrahydrofuran solution to 100 mL to get DDH solution (0.06 mmol L −1 ). 1.0 mg abamectin B 1 was added to a volumetric flask and mixed with tetrahydrofuran solution to 1 L to get abamectin B 1 solution (1.0 mg L −1 ). The appropriate excitation wavelengths and emission wavelengths of TPB in the absence and existence abamectin B 1 and DDH were analyzed by fluorescent spectra respectively. One 10 mL colorimetric tube was filled with probe TPB (1.0 mL, 0.06 mmol L −1 ) and avermectin B 1 (0.2 mL, 1.0 mg L −1 ). The other was only filled with probe TPB (1.0 mL, 0.06 mmol L −1 ). One 10 mL was only filled with DDH (1.0 mL, 0.06 mmol L −1 ). Then, they all fixed with tetrahydrofuran to 10 mL. For each sample in colorimetric tubes, test condition of the fluorescence intensity was as follows: sample mixed time: 10 s, Sampling interval: 5 nm, Scan speed: 12,000 nm min −1 , EX Slit: 5 nm, EM Slit: 5 nm, PMT Voltage: 700 V, Contour interval: 10 nm, Temperature: room temperature, EX WL: 200-600 nm, EM WL: 200-600 nm.
In order to explore the quantitative relationship between TPB and avermectin. The fluorescence intensity of the reacted product at 420 nm were examined at different concentrations of abamectin B 1 (0.00, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12 mg L −1 ). Test condition of the fluorescence intensity was same as that of probe TPB, except EX WL: 360 nm and EM WL: 200-700 nm. considering the emergence of esterification at pH below 5.0 and salt forming at pH above 5.0 for avermectin B 1 , the effect of pH 5.0-9.0 on the analysis result was investigated. Five 10 mL colorimetric tubes were filled with probe TPB (1.0 mL, 0.06 mmol L −1 ) and avermectin B 1 (0.2 mL, 1.0 mg L −1 ), and fixed to 10 mL with pH 5.0-9.0 phosphate buffer (0.2 M) at room temperature, respectively. For each sample in colorimetric tubes, test condition of the fluorescence intensity was same as that of probe TPB, except EX WL: 360 nm and EM WL: 420 nm. pH 6.0 phosphate buffer (0.2 M) was found as an ideal pH condition and used in following tests.
To obtain the ideal mount of phosphate buffer, the effect of 0.2-1.2 mL phosphate buffer (0.2 M, pH 6.0) was investigated. Six 10 mL colorimetric tubes were filled with probe TPB (1.0 mL, 0.06 mmol L −1 ) and avermectin B 1 (0.2 mL, 1.0 mg L −1 ), and fixed with 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mL phosphate buffer (0.2 M, pH 6.0) at room temperature, respectively. For each sample in colorimetric tubes, test condition of the fluorescence intensity was same as that of probe TPB, except EX WL: 360 nm and EM WL: 420 nm. 0.8 mL phosphate buffer (0.2 M, pH 6.0) was found as an ideal mount and further applied in followed tests.
To validate the method under analytical control, the limit of detection (LOD), limit of quantification (LOQ), precision and linear range were implemented according to the previous methods 21,22 . A 10 mL colorimetric tubes was mixed with abamectin B 1 (0.20 mL, 1.0 mg L −1 ) and probe TPB (1.0 mL, 0.06 mmol L −1 ), mixed with 0.8 mL phosphate buffer (0.2 M, pH 6.0), fixed with tetrahydrofuran to 10 mL and fluorescently detected according to that of probe TPB, except EX WL: 360 nm and EM WL: 420 nm. Eleven groups of parallel experiments were conducted. LOD and LOQ were calculated using the formulas shown in Eqs. (1) -(2). Precision was evaluated using relative standard deviation (RSD). The linear range was from LOQ to the maximum measured value.  Preparation and analysis of apple samples. Malus pumila mill, Qinguan and Huangxiangjiao were purchased from local Xingfu Supermarket (Beijing, China) and analyzed. Apple samples were prepared according to a published method 23 . Each sample (20.0 g) was respectively added with acetonitrile (10.0 mL) and vigorously shaken for 2.0 min by a vortex mixer. Then, each mixture was mixed with 4.0 g anhydrous magnesium sulfate anhydrous and 1.0 g sodium chloride and shook for another 1.0 min. Following centrifugation at 4000 rpm for 5.0 min, 2.0 mL of the upper layer was transferred to a 20 mL volumetric flask and filled with tetrahydrofuran to obtain tested Malus pumila mill, Qinguan and Huangxiangjiao samples. These samples were fluorescently detected according to that of probe TPB, except EX WL: 360 nm and EM WL: 420 nm, respectively. Value of abamectin B 1 was calculated according to the linear regression equation of this work.