Photoresponsive Surface Molecularly Imprinted Polymers for the Detection of Profenofos in Tomato and Mangosteen

As a moderately toxic organophosphorus pesticide, profenofos (PFF) is widely used in agricultural practice, resulting in the accumulation of a high amount of PFF in agricultural products and the environment. This will inevitably damage our health. Therefore, it is important to establish a convenient and sensitive method for the detection of PFF. This paper reports a photoresponsive surface-imprinted polymer based on poly(styrene-co-methyl acrylic acid) (PS-co-PMAA@PSMIPs) for the detection of PFF by using carboxyl-capped polystyrene microspheres (PS-co-PMAA), PFF, 4-((4-(methacryloyloxy)phenyl)diazenyl) benzoic acid, and triethanolamine trimethacrylate as the substrate, template, functional monomer, and cross-linker, respectively. PS-co-PMAA@PSMIP shows good photoresponsive properties in DMSO/H2O (3:1, v/v). Its photoisomerization rate constant exhibits a good linear relationship with PFF concentration in the range of 0~15 μmol/L. PS-co-PMAA@PSMIP was applied for the determination of PFF in spiked tomato and mangosteen with good recoveries ranging in 94.4–102.4%.


INTRODUCTION
Profenofos (PFF), [O-(4-bromo-2-chlorophenyl) O-ethyl S-propyl phosphorothioate], is widely used to control pest strains that are resistant to chlorpyrifos and other organophosphorus pesticides in fruit trees, vegetables, and cotton (He et al., 2010;Dadson et al., 2013). However, PFF is moderately to highly toxic to animals with an LD50 of 500 mg/kg (Gotoh et al., 2001;Ma et al., 2019). Thus, it is essential to detect PFF in agricultural products. Common detection methods for PFF include gas chromatography (Yang et al., 2012), high-performance liquid chromatography (HPLC) (Raharjo et al., 2009), biosensors Selvolini et al., 2018;Xiong et al., 2018), and colorimetric and fluorescent sensors (Zhang et al., 2014;Li X. et al., 2018;Kovida and Koner, 2020). Although those methods are sensitive, they are time-and reagent-consuming and require a professional operator, and sample preparation is complex. Furthermore, most of them demand expensive instruments.
Molecularly imprinted polymers (MIPs) are synthesized by copolymerizing functional monomers and cross-linkers in the presence of template molecules. The elution of template molecules makes it possible for the formation of tailor-made recognition cavities complementary to the template molecule in shape, size, and chemical functionality in the highly crosslinked polymer matrix. Meanwhile, those imprinted cavities are endowed with the established ability to recognize the target analyte, i.e., template molecules (Xu et al., 2013). MIPs are widely applied in purification and separation (Li et al., 2015;Gong et al., 2017a;He et al., 2018;Zhang et al., 2018;Gomez-Arribas et al., 2019), chiral recognition (Rutkowska et al., 2018;Zhao et al., 2019;Li et al., 2020), chemo-and bio-sensing (Altintas et al., 2016;Gong et al., 2017b;Ding et al., 2020;Kalecki et al., 2020), and catalysis and degradation (Liu et al., 2015;Zheng et al., 2017;Boitard et al., 2019;Muratsugu et al., 2020) due to its physical stability, thermal stability, low cost, and ease of preparation.
Stimulus-responsive molecularly imprinted polymers are generally prepared by introduction of stimulus-responsive moieties into MIPs. These MIP materials can respond to specific external stimuli such as temperature, light, magnetic field, solvent composition or solvent polarity, pH, or electric field through altering itself greatly at solubility, molecular chain structure, surface structure, and swelling or dissociation behavior. Among them, photoresponsive MIPs (PMIPs) attract great research attention because of the advantages of clean, remote control and no damage to sample (Schumers et al., 2010). PMIPs make it possible for template extraction, recognition, and release under external photo-irradiation at a certain wavelength (Alaei et al., 2018). The photoresponsive azobenzene moiety is generally introduced into MIP/SMIP by using azobenzene derivatives as a functional monomer (Gong et al., 2006. Good achievements have been obtained in PMIPs in recent years. Azobenzene derivatives containing hydrophilic -SO 3 H and -COOH groups were designed as the functional monomers to realize the photo-response of PMIPs in aqueous solution (Tang et al., 2012. The o-methyl and o-methoxy azobenzene derivatives were designed to realize visible-light response for both trans to cis and cis to trans photoisomerization Gong et al., 2019). Photoresponsive SMIPs (PSMIPs) and hollow MIPs were fabricated to solve the problem of template being embedded in traditional PMIPs (Gong et al., , 2017cYang et al., 2018). PSMIPs have potential applications in drug delivery, electrochemical sensor, and environmental pollutant detection. However, the application in detecting pesticide residues by PMIPs or PSMIPs has been rarely reported.
In this article, we focused on developing a method to detect trace PFF in agricultural products such as mangosteen and tomato by using a photoresponsive surface molecularly imprinted polymer PS-co-PMAA@PSMIP. In the presence of the PFF template, MPABA and TEAMA were copolymerized on the surface of PS-co-PMAA by using potassium persulfate (KPS) as the initiator. PS-co-PMAA@PSMIPs were obtained after the subsequent removal of the PFF template, and specific recognition sites with memory of the shape, size, and functionality of PFF template were formed within PS-co-PMAA@PSMIPs. PS-co-PMAA@PSMIPs were applied to detect trace PFF in complex vegetable and fruit sample matrices. PS-co-PMAA@PSMIPs have a good binding capacity for PFF, a quick mass transfer rate, and excellent cycling stability on photoregulated release and uptake of PFF.

Synthesis of Functional Monomer, PS-co-PMAA Substrate, and Cross-Linker
The functional monomer MPABA was synthesized according to the method reported by Gong et al. (2006) by using p-aminobenzoic acid as the starting material. The cross-linker TEAMA was synthesized according to the method reported by Li et al. (2015) by using methacrylic acid, thionyl chloride, and triethanolamine as the starting materials. PS-co-PMAA substrate was synthesized according to the method reported by Yang et al. (2018) by using styrene and methacrylic acid as the raw materials.

Synthesis of PS-co-PMAA@PSMIPs and PS-co-PMAA@PSNIPs
PS-co-PMAA (50.00 mg) was dispersed in 10 mL DMSO/H 2 O (3:1, v/v) solution in a 100-mL three-necked bottle, sonicated for 30 min, and then stirred for 2 h to ensure the formation of stable suspension. MPABA (31.0 mg, 0.10 mmol) and PFF (7.5 mg, 0.02 mmol) were dissolved in a mixed solvent of DMSO/H 2 O (10 mL; 3:1, v/v), which was slowly added to the PS-co-PMAA suspension. The suspension was stirred for 12 h to ensure a complete interaction between MPABA and PFF through hydrogen bond. Subsequently, ∼5 mL TEAMA solution (70.7 mg, 0.20 mmol) in DMSO/H 2 O (3:1, v/v) was added by a syringe. The mixture was stirred for 30 min and evacuated for 10 min, and then nitrogen gas was bubbled below the liquid level of the suspension for 10 min to remove oxygen. During this process, 5 mL KPS solution (14.0 mg KPS was dissolved in 5 mL distilled water) was added. The mixture was placed in an oil bath at 80 • C for 24 h in a nitrogen atmosphere. After being cooled to room temperature, the product was separated by centrifuging at 10,000 rpm for 5 min, washed with ethanol and water (1:1, v/v) until the supernatant was colorless, dried at 50 • C, and ground to powder. This powder was further Soxhlet-extracted with methanol and acetic acid (4:1, v/v) solution for 48 h and then Soxhlet-extracted with methanol for 24 h to fully elute the PFF. Finally, the eluted powder was dried at 50 • C to obtain PS-co-PMAA@PSMIPs (123.2 mg). PS-co-PMAA@PSNIPs were synthesized using the same way but without the addition of template PFF.

Spectroscopic Characterization and Photoisomerization Studies
Spectroscopic characterization and photoisomerization of MPABA and PS-co-PMAA@PSMIPs were performed in DMSO/H 2 O (3:1, v/v) according to the method reported by Gong et al. (2016). The photoisomerization kinetic rate constants (k) from trans to cis and cis to trans are calculated according to Equation 1, where A 0 , A t , and A ∞ are the absorbance of the azobenzene chromophores at their corresponding wavelengths at times 0 and t and at the photo-stationary stage, respectively, and k is the rate constant of the photoisomerization process.

Binding Kinetics of PS-co-PMAA@PSMIPs and PS-co-PMAA@PSNIPs for PFF
The adsorption kinetics of PS-co-PMAA@PSMIPs was studied. Approximately 5 mL PFF solution (300 µmol/L) in DMSO/H 2 O (3:1, v/v) and 20.0 mg PS-co-PMAA@PSMIPs were placed in each plastic centrifuge tube, sealed, and dispersed ultrasonically evenly. The obtained suspensions were incubated for different predetermined times (5, 15, 30, 45, 60, 90, 120, and 240 min, respectively) in the dark and then centrifuged at 10,000 rpm for 5 min. The supernatant was filtered through a 0.22-µm polyethersulfone syringe filter and then analyzed by UV-Vis spectrophotometry. The adsorption kinetics experiment of PSco-PMAA@PSNIPs was measured using the same method. All experiments were repeated three times, and the averaged value was used. The binding capacity was calculated according to Equation 2 (Long et al., 2016), where C 0 and C s represent the initial and equilibrium concentrations of PFF in solution (mg mL −1 ), V is the volume of the bulk solution (mL), and m is the mass (g) of the material.

Adsorption Isotherms of PS-co-PMAA@PSMIPs/PS-co-PMAA@PSNIPs for PFF
Approximately 20.0 mg of PS-co-PMAA@PSMIPs or PS-co-PMAA@PSNIPs was suspended in 5-mL PFF solutions in   DMSO/H 2 O (3:1, v/v) with various PFF concentrations, ultrasonically dispersed evenly, and sealed. The obtained suspension was incubated for 2 h in the dark and then centrifuged at 10,000 rpm for 5 min. The supernatant was filtered through a 0.22-µm polyethersulfone syringe filter and then measured by UV-Vis spectrophotometry. All experiments were repeated three times, and the averaged value was used. The adsorption kinetic capacity of PS-co-PMAA@PSMIPs for PFF (Q) was calculated according to Equation 3: where Q max , Q, C s , and K d represent the maximal chemical binding capacity of PS-co-PMAA@PSMIPs for PFF (mg/g), the equilibrium adsorption capacity of PFF bound to polymers (mg/g), the equilibrium concentration of PFF in solution (mg/L), and the dissociation constant (mg/L). The binding specificity of PS-co-PMAA@PSMIPs and PS-co-PMAA@PSNIPs was investigated by using the structural analogs CPF and CPFm as reference compounds. The specific research method is the same as described above, but 5 mL CPF or CPFm solution (300 µmol/L) in DMSO/H 2 O (3:1, v/v) was used.

Photoregulated Release and Uptake Studies
For the purpose of studying the photoregulated release and uptake, 20.0 mg of PS-co-PMAA@PSMIPs or PS-co-PMAA@PSNIPs was suspended in 5 mL PFF solution (300 µmol/L) in DMSO/H 2 O (3:1, v/v). The obtained suspension was ultrasonically dispersed, sealed, and incubated for 2 h in the dark and centrifuged at 10,000 rpm for 5 min. The supernatant was filtered through a 0.22-µm polyethersulfone syringe filter and then measured by UV-Vis spectrophotometry. The adsorption capacity of PS-co-PMAA@PSMIPs or PS-co-PMAA@PSNIPs to PFF was calculated. PS-co-PMAA@PSMIPs or PS-co-PMAA@PSNIPs were re-dispersed in the supernatant to form a uniform suspension and then alternately irradiated at 365 nm for 100 min and 440 nm for 45 min. In the interval of alternating irradiation, the adsorption capacity of PS-co-PMAA@PSMIPs or PS-co-PMAA@PSNIPs to PFF was measured and calculated in the same way as described above.

Determination of PFF in Tomato and Mangosteen
Approximately 20.00 g of the homogeneous tomato or mangosteen sample was added to two 50-mL plastic centrifuge tubes, to which 0.1 and 0.25 mg standard samples of PFF were, respectively, added. After incubating for 4 h, the spiked samples were extracted ultrasonically with 15 mL DMSO/H 2 O (3:1, v/v) solution for about 20 min. The above extraction was repeated two times. The mixture was centrifuged at 12,000 r/min for 5 min. The supernatant was subsequently filtered through a 0.22-µm polyethersulfone syringe filter, transferred into a 50-mL volumetric flask, and diluted to the mark with DMSO/H 2 O (3:1, v/v).

Characterization of PS-co-PSMIPs and PS-co-PSNIPs
As shown in Figure 2, PS-co-PMAA with a smooth surface has a narrow distribution of 215-225 nm and the particle size is mainly about 218 nm in diameter, while the polymer particles PS-co-PMAA@PSMIPs and PS-co-PMAA@PSNIPs (Supplementary Figure 1) have a rough surface with a distribution of 265-295 nm and the particle size is mainly about 280 nm in diameter. This indicates that the copolymer layer of MPABA and TEAMA was uniformly grown on the

FIGURE 6 | The relationship between binding capacity of PS-co-PMAA@PSMIPs/PS-co-PMAA@PSNIPs and binding time for PFF.
substrate surface with a thickness of ∼30 nm, since the carboxylmodified polystyrene microspheres have good compatibility with the copolymer layer, which is conducive to the formation of a uniform shell layer on polystyrene matrices. As shown in Figure 3, in the polymer particles of PS-co-PMAA@PSMIPs and PS-co-PMAA@PSNIPs, the out-of-plane C-H bending vibration peaks of MPABA at 756 cm −1 and 699 cm −1 and the benzene-ring carbon skeleton stretching vibration peaks from PS-co-PMAA at 1,590 and 1,495 cm −1 are retained , and the stretching vibration peak at 1,737 cm −1 owing to C=O from cross-linkers is enhanced (Wang et al., 2019). This indicated that the monomer and the cross-linkers were successfully copolymerized on PS-co-PMAA.
The porous nature of PS-co-PMAA@PSMIPs and PS-co-PMAA@PSNIPs was investigated by N 2 adsorption-desorption analysis. As shown in Figure 4, the isotherms of PS-co-PMAA@PSMIPs and PS-co-PMAA@PSNIPs approached to type IV isotherms. The analysis results of the BET method for surface areas and the BJH method for pore volume are summarized in Table 1. PS-co-PMAA@PSMIPs had larger values in surface area, pore volume, and pore size owing to imprinted cavities.

Photoisomerization Analysis
The spectroscopic responses of PS-co-PMAA@PSMIPs were consistent with MPABA (Figures 5A-D). This indicated that photoresponsive properties of azobenzene chromophores were successfully retained in PS-co-PMAA@PSMIPs, while the photoisomerization ability declined slightly since the rotation of azobenzene was prevented by a rigid three-dimensional imprinted copolymer layer ( Table 2). The trans→ cis and cis→ trans photoisomerization rate constants of PS-co-PMAA@PSMIPs were 1.98-and 1.02-fold smaller than those of MPABA. The trend was consistent with previous reports (Gong et al., 2006Li et al., 2015). Compared with the trans→ cis photoisomerization rate constant, the possible reason for the smaller decrease in cis→ trans photoisomerization rate constant was that sufficient free volume was created after trans→ cis photoisomerization. Nonetheless, under alternating irradiation at 365 and 440 nm, the photoisomerization reversibility of PS-co-PMAA@PSMIPs did not significantly decrease after five cycles (Figures 5E,F).

Binding Kinetics
In DMSO/H 2 O (3:1, v/v), the adsorption intensity of PFF at 277 nm increased as its concentration increased (Supplementary Figure 3A), and a good linear relationship was observed between the absorbance at 277 nm and PFF concentration (Supplementary Figure 3B). Therefore, the absorbance at 277 nm was used to estimate the PFF concentration in the corresponding solution by using Supplementary Figure 3B as the standard curve. As shown in Supplementary Figure 4, the absorbance at 277 nm decreased as the incubation time increased; this illustrates that PFF in the solution was gradually absorbed by PS-co-PMAA@PSMIPs. An equilibrium was obtained after 60 min. Based on these data, the binding capacity of PS-co-PMAA@PSMIPs/PS-co-PMAA@VPSNIPs toward PFF was calculated using Equation 2. It is seen from Figure 6 and Supplementary Table 1 that the binding capacity of PS-co-PMAA@PSMIPs/PS-co-PMAA@VPSNIPs toward PFF shows a trend of increasing first within 60 min and then stabilizing. The binding capacity of PS-co-PMAA@PSMIPs (10.56 mg/g) was significantly higher than the PS-co-PMAA@VPSNIP material (4.38 mg/g) owing  to the imprinted cavities. PS-co-PMAA@PSMIPs reached the adsorption equilibrium in about 45 min, while PS-co-PMAA@VPSNIPs took about 90 min. This was ascribed to the strong affinity of the imprinted cavities that made the template quickly bind to PS-co-PMAA@PSMIPs.
As shown in Figure 8, the isothermal adsorption data of PS-co-PMAA@PSMIPs was analyzed by the Scatchard model. Two straight lines with different slopes were observed, which indicated that there were two types of binding sites on  PS-co-PMAA@PSMIPs for PFF, namely, high-affinity sites and low-affinity sites. The steeper straight line originated from the strong affinity of the imprinted cavities to PFF, from which K d was calculated as 61.35 mg/L, and Q max was 14.98 mg/g, while the flatter line showed the weak affinity binding capacity owing to physical adsorption on the surface of PS-co-PMAA@PSMIPs. PSco-PMAA@PSNIPs had only a weak physical binding property due to the absence of the imprinted cavities of PFF.

Binding Selectivity
The binding capacity of PS-co-PMAA@PSMIPs to PFF,CPFm,and CPF was,respectively about 39.7,8.3,and 7.6% (Figure 9). The binding capacities of PS-co-PMAA@PSMIPs to PFF were, respectively, 4.8-and 5.2-fold larger than those of PFF CPFm and CPF. The difference in binding capacity came from the matching degree of the imprinted cavities, shape, and size to the binding molecule. The above results indicated that PS-co-PMAA@PSMIPs had a specific binding capacity to PFF, while the binding capacity of PS-co-PMAA@PSNIPs to PFF,CPFm,and CPF was,respectively about 13.0,7.4,and 7.0%. This demonstrated that PS-co-PMAA@PSNIPs only had nonspecific binding capacity to PFF.

Photoregulated Release and Uptake of PFF
The photoregulated release and uptake of PS-co-PMAA@PSMIPs for PFF were investigated under alternating irradiation at 365 and 440 nm in DMSO/H 2 O (3:1, v/v) solution. The process was repeated five times. As shown in Figure 10, when the material reached the adsorption equilibrium in the dark, imprinting cavities on PS-co-PMAA@PSMIPs were fully filled with PFF. Under irradiation at 365 nm, the azobenzene chromophore in PS-co-PMAA@PSMIPs underwent a configuration change from trans to cis. During this process, the shape and size of the PFF-imprinted cavities changed; this caused the PFF bound in the imprinted cavities to be squeezed or loosened and then fall from the cavities into the solution. Therefore, the binding capacity of PS-co-PMAA@PSMIPs for PFF was reduced from 11.06 mg/g to 5.05 mg/g. Upon irradiation at 440 nm, the azobenzene chromophore in PS-co-PMAA@PSMIPs underwent a configuration change from cis to trans, which caused the PFFimprinted cavities to return to its original shape and size. During this process, PFF was rebound in the imprinted cavities due to the affinity of the cavities. So the binding capacity of PS-co-PMAA@PSMIPs for PFF was increased from 5.05 to 11.04 mg/g. During a repeating in the photo-switching cycle, the release and uptake of PFF were similar to the previous cycle. This excellent capacity, however, was not working on PS-co-PMAA@PSNIPs because of its non-imprinted cavities.

Determination of Trace PFF in Spiked Mangosteen and Tomato Samples
As shown in Figure 11, the imprinted cavities inside the PS-co-PMAA@PSMIPs were gradually filled as the PFF concentration increased, and the photoisomerization rate constant (k) of PSco-PMAA@PSMIPs decreased firstly. As the PFF concentration increased to 20 µmol/L, k tended to be invariant. When the concentration of PFF reached a certain level, the imprinted cavities of PS-co-PMAA@PSMIPs were filled with PFF. There was insufficient free volume for azobenzene chromophore of the imprinted materials to rotate, thus making isomerization more difficult. k showed a good linear relationship with the PFF concentration in the range of 0∼15 µmol/L. The detection limit was 0.40 µmol/L (0.35 mg/kg), which is below the maximum residue limit (10 mg/kg) for PFF in mangosteen and tomato samples stipulated by the National Standards of the People's Republic of China (GB-2763-2019). The feasibility of the test method was evaluated by detecting trace PFF in mangosteen or tomato samples at two spiked concentrations (2.00 mg/L, 5.00 mg/L). As shown in Table 3, the recovery data for spiked PFF standards ranged from 94.4 to 102.4 and relative standard deviation (RSD%) ranged from 2.4 to 5.9%. Therefore, the concentration of PFF in the detection system can be calculated by measuring the photoisomerization rate constant of PS-co-PMAA@PSMIPs. Hence, a convenient method for determining trace PFF by ultraviolet light response was established.
In comparison with reported methods for detecting PFF (Raharjo et al., 2009;Yang et al., 2012;Zhang et al., 2014;, the method in this work had a higher detection limit (Table 4). However, this value (0.35 mg/kg) is below the maximum residue limit (10 mg/kg) for PFF in mangosteen and tomato samples stipulated by the National Standards of the People's Republic of China (GB-2763-2019) and can satisfy the requirement in this field. It is noteworthy that the sample preparation is simple, and expensive instruments or special materials (e.g., biomaterials) are required.

CONCLUSION
In summary, photoresponsive surface molecularly imprinted polymers (PS-co-PMAA@PSMIPs) with a uniform shell thickness were prepared by combining stimulus-responsive polymers with surface molecular imprinting technology. The hydrogen bond driving effect from the PS-co-PMAA surface and MPABA and the good compatibility between the PSco-PMAA-based organic substrate and the copolymer layer are all conducive to the formation of a homogeneous shell polymer, which contributed to the binding capacity of PS-co-PMAA@PSMIPs. The photoresponsive properties of azobenzene chromophore were well-retained. PS-co-PMAA@PSMIPs can specifically bind PFF. PS-co-PMAA@PSMIPs were applied to detect trace PFF in fruit and vegetable samples with complex matrices with good recoveries and low relative standard deviation.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.

AUTHOR CONTRIBUTIONS
MC performed the experiments and wrote the draft. HY and YS performed parts of the experiments. QT provided some ideas, discussed, and revised the manuscript. CC provided some ideas and discussed. CG designed, supervised, and revised the manuscript. All authors contributed to the article and approved the submitted version.