A signal-on detection of organophosphorus pesticides by fluorescent probe based on aggregation-induced emission

A signal-on detection of organophosphorus pesticides by fluorescent probe based on aggregation-induced emission

Sensors & Actuators: B. Chemical 292 (2019) 156–163 Contents lists available at ScienceDirect Sensors and Actuators B: Chemical journal homepage: ww...

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Sensors & Actuators: B. Chemical 292 (2019) 156–163

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

A signal-on detection of organophosphorus pesticides by fluorescent probe based on aggregation-induced emission ⁎⁎

Yue Caia,1, Jingkun Fangd,1, Bingfeng Wanga, Fangshuai Zhangb,c, Guang Shaob,c, , Yingju Liua,

T



a

College of Materials & Energy, South China Agricultural University, Guangzhou, 510642, China School of Chemistry, Sun Yat-sen University, Guangzhou, 510275, China c Shenzhen Research Institute, Sun Yat-sen University, Shenzhen, 518057, China d School of Chemical Engineering, Nanjing University of Science and Technology, Nanjing, 210094, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Organophosphorus pesticides Aggregation-induced emission Acetylcholinesterase Tetraphenylethene

Organophosphorus pesticides (OPs) have been broadly used in agriculture because of their high insecticidal efficiency. However, most OPs are highly toxic, since they can irreversibly inhibit acetylcholinesterase (AChE), leading to the neurotransmitter acetylcholine disorder which triggers a series of neurological diseases, and even death. Therefore, the rapid and sensitive detection of OPs is of great significance to life health and environmental protection. In this work, new derivatives of tetraphenylethene (TPE) with one or two aldehyde groups were synthesized and characterized, showing that they exhibited different aggregation-induced emission (AIE) effects in different pH solutions. Especially, the hydrolysis product of acetylthiocholine iodide (ATCh) catalyzed by acetylcholinesterase (AChE) can influence the pH of solution, causing the protonation of TPE-1 and changing its fluorescent intensity. Considering that the AChE activity can be specifically inhibited by OPs, the proposed TPE-1 was used to detect OPs between 0.009 and 22.5 mg/L with a detection limit of 0.008 mg/L. In addition, the selectivity was acceptable, providing a possible application for the detection of OPs.

1. Introduction With the improvement of people's living standards, food safety has received more and more attention from the public. It is well known that a number of harmful substances are difficult to degrade in a short period of time under natural conditions, resulting in detrimental residuals in agricultural products. At present, many pesticides including various organophosphorus, triazine and pyrethroids have been used to prevent pests and diseases of crops. Nevertheless, the large and longterm use of pesticides can cause serious pollution of air, water, soil and agricultural products, and ultimately endanger ecosystems including humans. Due to the significantly effect of the eradication of pests, organophosphorus pesticides (OPs) are one of the most used pesticides [1,2]. However, as a neurotoxin and irreversible inhibition of the activity of cholinesterase, the unreasonable use of OPs can cause a large accumulation of acetylcholine in the human body, thus neurological diseases including Parkinson's disease, schizophrenia and Alzheimer's disease, or even fatal consequences can be found [3–8]. In this regard, the construction of a rapid, sensitive and reliable method for OPs

detection has important and far-reaching significance, related not only to food safety and human health, but also to the ecosystem protection. To date, many kinds of analytical methods including gas chromatography/mass spectrometry (GC/MS) [9], electrochemical analysis [10,11], and high-performance liquid chromatography (HPLC) [12] have been developed to detect OPs. Regrettably, these methods still remain several disadvantages, such as expensive instrument, timeconsuming and complicate operation. In recent years, a fluorescencebased strategy has drawn more and more attention due to its high sensitivity and cost-efficiency. Fluorescence possesses the unique luminescence characteristics which can offer a promising analytical platform by reducing background signals greatly and improving sensitivity strikingly through the combination of photo-excitation-emission [13–16]. Consequently, several fluorescence detection strategies have been reported. For example, Xiao et al used MoOx quantum dots to detect the activity of acetylcholine esterase and its inhibitors [17], while Hou et al used quaternized carbon dots to develop a fluorescence resonance energy transfer method for dichlorvos detection [18] and Wang el al reported a novel non-enzymatic method to detect malathion



Corresponding author. Corresponding author at: School of Chemistry, Sun Yat-sen University, Guangzhou, 510275, China. E-mail addresses: [email protected] (G. Shao), [email protected] (Y. Liu). 1 The authors are equally contributed to this work. ⁎⁎

https://doi.org/10.1016/j.snb.2019.04.123 Received 16 January 2019; Received in revised form 18 April 2019; Accepted 25 April 2019 Available online 25 April 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.

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Scheme 1. Synthesis of TPE-1 and TPE-2.

properties of AIE fluorophores in bioassays, especially for the detection of OPs [33–36]. In this work, we synthesized TPE derivatives, characterized their structure and detected pH sensitively based on their AIE effect. In the presence of AChE, acetylthiocholine iodide (ATCh) can be hydrolyzed to produce acetic acid, changing the pH of the solution and thus influencing the fluorescence. Finally, the enzymatic ability of AChE can be inhibited by OPs, thus the fluorescence detection of OPs can be indirectly realized.

in water [19]. In addition, enzyme-based assays are also used to detect OPs, based on the indirect detection of pesticides from the inhibition of acetylcholinesterase (AChE) activity [20,21]. Among the enzyme assays, AChE is a cholinesterase with carboxypeptidase and aminopeptidase activity, which can catalyze the hydrolysis of acetylcholine selectively. OPs can be phosphorylated with the hydroxyl group of serine on AChE to form stable covalent bonds, which hinders the reaction of AChE with the amino groups with the substrate choline, and reduces the activity of cholinesterase [22]. However, the effective design of such OPs fluorescence sensors was restricted due to the fluorescence quench from the substrate or the unstable probes [23]. As you know, most organic fluorescence molecules suffer from aggregation-caused quenching (ACQ), whose fluorescence efficiency is greatly reduced at high concentration or solid state. The ACQ effect forces many molecules to restrict within narrow practical applications. Especially in bioassays, the degree of labeling which belongs to biological analytes by fluorescent molecules is significantly limited, forcing researchers to use dilute solutions of probes for biosensor applications, which results in dramatical sensitivity degradation and poses a huge obstacle to the tracking and analysis of biomolecules [24,25]. Hence, it seems to be the Achilles heel of organic luminescence materials. To weaken the ACQ effect, various physical, chemical, and engineering methods have been used to reduce the aggregation between molecules, but the effect is not ideal. Recently, Tang and co-workers discovered a group of fluorescent molecules with aggregation-induced emission (AIE) characteristics [26]. In comparison with conventional fluorophores, this kind of molecules exhibit strong fluorescence in the aggregation state. Besides, they have demonstrated that AIE phenomenon is owing to the restriction of intramolecular motion [27]. Its unique properties provide significant advantages for AIE probes such as lower background and higher light stability [28]. After that, many research groups have devoted on designing new AIE molecules and discussing their applications in several fields [29]. Among them, tetraphenylethene (TPE) is a representative molecule, which has been applied in bioanalysis by modifying various groups [30–32]. Despite this, there are few reports on the pH-sensitive

2. Experimental 2.1. Reagents AChE and ATCh were purchased from Aladdin Regent Co., Ltd. Palladium acetate, 4,4-dibromobenzophenone, 4,4-dimethoxydiphenylamine, sodium tert-butoxide and other solvents and salts were obtained from Sinopharm Chemical Reagent Co., Ltd. Dimethoate, emamectin benzoate, spirotetramat, hexythiazox, tebuconazole and fenpyroximate were obtained from Dr. Jinliang Jia, South China Agricultural University. Except that tetrahydrofuran was re-steamed with sodium tablets and dried with 4A molecular sieves, other solvents and chemicals were used without further treatment.

2.2. Instruments The fluorescence measurements were performed by using an F-7000 fluorescence spectrometer (Hitachi, Japan) and an FLS-980 fluorescence spectrometer (Edinburgh, England). Nuclear magnetic resonance spectrometer spectrometer (NMR, Bruker 400 & VARIAN 300, Bruker & VARIAN, Germany & USA) and liquid chromatograph mass spectrometer (LC–MS, Uplc1290-6540B Q-TOF, Agilent, America) were used to characterize the structure of the substances. Dynamic light scattering (DLS) was performed on Zetasizer Nano ZSE (Malvern, England). 157

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55.43. MS (APCI) m/z: [M+H]+ Calcd for C64H51N2O6S2: 1007.3110; Found: 1007.3092.

2.3. Synthesis of AIE molecules 2.3.1. Synthesis of compound 1 As in Scheme 1, 4,4-dibromobenzophenone (0.5 mmol, 170 mg, 1 eq), 4,4-dimethoxydiphenylamine (1.25 mmol, 286 mg, 2.5 eq), palladium acetate (0.025 mmol, 5.6 mg, 0.05 eq), tri-tert-butylphosphine (0.0375 mmol, 7.58 mg, 0.075 eq), sodium tert-butoxide (3 mmol, 288.3 mg, 6 eq) and re-steamed toluene (5 mL) were added to the flask with an argon atmosphere. Afterwards, it was transferred to an oil bath and heated at 130 °C for 24 h. The crude product was purified using petroleum ether/CH2Cl2/ethyl acetate (2:1:0.05, v/v/v) as the eluent on silica gel column (286 mg, 89%). 1H NMR (400 MHz, CDCl3, Fig. S1) δ 7.63 (d, J = 8.9 Hz, 4H), 7.12 (d, J = 8.9 Hz, 8H), 6.88–6.83 (m, 12H), 3.81 (s, 12H). The data of the 1H NMR are in keeping with the reported ones [37].

2.4. Sensitive fluorescence detection of pH The stock solutions of TPE-1 and TPE-2 (1 mM) were prepared in DMF. This solution was further diluted to 100 μM by DMF/H2O (1:4, v:v) for fluorescence detection, while the buffers were PBS with different pH values including 3.20, 3.56, 4.12, 4.98, 5.29, 5.91, 6.47, 6.81, 7.17, 7.38 and 7.71. To investigate the possible mechanism for pH response, the DFT calculation at B3LYP/6-31+G(d) level of the Gaussian 09 packagewas employed by using the CPCM solvation model to compute solvent effects in DMF [38]. Starting with the optimum ground structures, the geometry of the excited state potential energy surfaces was optimized by the time-dependent DFT calculations to simulate the fluorescence. At TD B3LYP/6-31+G(d) level, the transition characteristics of fluorescence emissions were calculated.

2.3.2. Synthesis of compound 2 Zinc powder (15 mmol, 981 mg, 10 eq) and dried tetrahydrofuran were added to the double-mouth bottle under argon protection. Subsequently, TiCl4 (7.5 mmol, 0.83 mL, 5 eq) was added to the bottle at −10 °C, stirred for 30 min at room temperature and then refluxed in oil bath at 85 °C. After 3 h, pyridine (7.5 mmol, 0.6 mL, 5 eq) was added to the bottle at −10 °C and stirred for another 10 min. Then, compound 1 (1.5 mmol, 955 mg, 1 eq) and 4,4-dibromobenzophenone (1.8 mmol, 612 mg, 1.2 eq) were dissolved in re-distilled tetrahydrofuran, added and refluxed for 13 h at 85 °C. After the reaction, saturate K2CO3 solution was poured into the mixture and then extracted with ethyl acetate. After vacuum rotary evaporation, the residue was purified by using petroleum ether/CH2Cl2 (1:1, v/v) as an eluent on silica gel column (383 mg, 27%). 1H NMR (400 MHz, CDCl3, Fig. S2) δ 7.24 (d, J = 8.0 Hz, 4H), 7.00 (d, J = 8.0 Hz, 8H), 6.88 (d, J = 8.0 Hz, 4H), 6.81-6.78 (m, 12 H), 6.65 (d, J = 8.7 Hz, 4H), 3.78 (s, 12 H). 13C NMR (101 MHz, CDCl3, Fig. S3) δ 155.77, 147.36, 143.09, 142.29, 140.69, 136.02, 134.87, 133.07, 132.05, 130.80, 126.52, 120.02, 119.37, 114.60, 55.46.

2.5. Organophosphorus pesticides (OPs) detection For AChE detection, different volumes of ATCh were added into 170 μL TPE-1 or TPE-2 in pH = 7.2 DMF/H2O (1:4, v:v) with 10 μL of 2.5 mU AChE. After reaction for 5 min, the corresponding fluorescence spectra were recorded. Then, dimethoate was selected as a model analyte to inhibit AChE activity. For OPs detection, 10 μL of 2.5 mU AChE was incubated with 30 μL of OPs solution with different concentrations for 15 min. Then, they were transformed to 170 μL of TPE-1 or TPE-2 in DMF/H2O (1:4, v:v), followed by adding 120 μL of 0.3 M ATCh and recording the fluorescence spectra. After that, the interferences of other pesticides were tested by incubating the same concentration of pesticides, while other conditions remained. Finally, the water sample of Poyang Lake of South China Agricultural University was selected as the real sample. After filtration, the filtrate was detected for different concentrations of dimethoate by standard addition method. 3. Results and discussion

2.3.3. Synthesis of TPE-1 and TPE-2 Compound 2 (0.79 mmol, 747.8 mg, 1eq), 5-formylthiophene-2boronic acid (2.37 mmol, 370 mg, 3 eq), Pd[(PPh)3]4 (0.079 mmol, 91.3 mg, 0.1 eq), K2CO3 (7.9 mmol, 1090 mg, 10 eq), THF (16 mL) and deionized water (4 mL) were added to a flask under argon atmosphere, reacted at 45 °C for 24 h and extracted with CH2Cl2. After vacuum rotary evaporation, the residue was purified by using petroleum ether/ CH2Cl2/ethyl acetate (1:1:0.1, v/v/v) as the eluent on silica gel column, thus TPE-1 (246 mg, 32%) and TPE-2 (405 mg, 51%) with different aldehyde groups were obtained. The molecular structures of TPE-1 and TPE-2 were characterized by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). TPE-1 (Figs. S4–S6): 1H NMR (400 MHz, CDCl3) δ 9.87 (s, 1H), 7.72 (d, J = 4.0 Hz, 1H), 7.44 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 4.0 Hz, 1H), 7.26 (d, J = 4.0 Hz, 2H), 7.07 (d, J = 8.0 Hz, 2H), 7.00 (t, J = 8.0 Hz, 8H), 6.92 (d, J = 8.0 Hz, 2H), 6.85–6.80 (m, 8H), 6.77 (d, J = 8.0 Hz, 4H), 6.67–6.64 (m, 4H) and 3.78–3.75 (m, 12H). 13C NMR (100 MHz, CDCl3) δ 182.67, 155.79, 155.78, 154.31, 147.46, 147.43, 145.79, 143.16, 142.76, 141.97, 140.66, 140.63, 137.50, 136.91, 136.23, 134.87, 134.81, 133.15, 132.20, 132.15, 130.86, 130.52, 126.55, 126.52, 125.57, 123.70, 120.07, 119.31, 114.60, 114.55, 55.46, 55.43. MS (APCI) m/z: [M+H]+ Calcd for C59H48BrN2O5S: 975.2389 and 977.2389; Found: 975.2352 and 977.2351. TPE-2 (Figs. S7–S9): 1H NMR (400 MHz, CDCl3) δ 9.88 (s, 2H), 7.73 (d, J = 4.0 Hz, 2H), 7.46 (d, J = 8.0 Hz, 4H), 7.38 (d, J = 4.0 Hz, 2H), 7.11 (d, J = 8.0 Hz, 4H), 7.00 (d, J = 8.0 Hz, 8H), 6.86 (d, J = 8.0 Hz, 4H), 6.77 (d, J = 8.0 Hz, 8H), 6.66 (d, J = 8.0 Hz, 4H), 3.75 (s, 12H). 13C NMR (100 MHz, CDCl3) δ 182.69, 155.80, 154.31, 147.53, 145.87, 143.23, 141.98, 140.99, 137.53, 136.45, 134.82, 132.29, 132.25, 130.57, 126.96, 125.63, 123.72, 119.24, 114.96,

3.1. The AIE properties of TPE-1 As in Fig. 1A, the fluorescence of TPE-1 was studied in a mixture of DMF/H2O with different H2O fractions (0%–90%). TPE-1 had almost no fluorescence in absolute DMF solution, but the emission at solid state was strong with quantum yield (ФF) as high as 66.65%. The fluorescence spectrum in the mixed solvent showed double peaks at the shortwavelength of 446 nm and the long-wavelength of 593 nm [39]. When the H2O fraction increased, the fluorescence gradually increased, suggesting TPE-1 was aggregated after the addition of H2O. Under the UV lamp, along with the increasing of H2O fraction the solution transformed from nonluminescence to orange light, which could be clearly observed by naked eyes, also supporting its AIE properties (Fig. 1B) [24]. When a certain amount of water was added, due to the physical constraint such as steric hindrance, the intramolecular rotation was limited to block the non-radiative decay pathway of the molecule from the excited state to the ground state transition, thus the radiation relaxation became the main transition mode, eventually leading to the strong fluorescence when they were in agglomerated state [40,41]. Besides, both TPE-1 and TPE-2 exhibited the solid-state emission under the UV illumination of 365 nm, and their fluorescence emission peaks were situated at 604 nm and 626 nm, respectively (Fig. S10). Compared with TPE-1, the electron-withdrawing group of Br atom on the TPE was replaced by a 5-formylthiophene group, so the push-pull electron effect of electrons in the whole molecule was stronger; meanwhile, the thiophene group expanded the conjugated system, so the fluorescence peak of TPE-2 was red-shifted [42]. Moreover, Tyndall scattering demonstrated that compared with 158

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Fig. 1. (A) Fluorescence spectra of 10 μM TPE-1 in DMF/H2O mixtures with different H2O fractions, λex = 365 nm. (B) Plots of fluorescence intensity at 446 nm and 593 nm versus H2O fractions (N = 3). Inset: photo images at different H2O fractions (0%, 20%, 40%, 60%, 80%, 90%). (C) Tyndall scattering of TPE-1 in absolute DMF, DMF with H2O fraction of 5% and 80%. (D) DLS of TPE-1 in DMF/H2O mixture with H2O fraction of 50%, 60%, 70%, 80% and 90%.

addition, the change of fluorescence intensity TPE-2 was only 53.3% of TPE-1, suggesting its sensitivity to pH was not as obvious as TPE-1. As a result, TPE-1 was used as a signal molecule in the subsequent experiments.

absolute DMF and DMF with 5% H2O fraction, the aggregates were formed in DMF solution with 80% H2O fraction (Fig. 1C). Then, dynamic light scattering (DLS) was used to characterize the size distribution of DMF/H2O mixture with water fraction of 0%, 50%, 60%, 70%, 80% and 90%. The DLS measurement showed that no aggregates were formed in pure DMF solution (not shown), but the aggregates with average particle sizes of 59.56 nm, 6814 nm, 79.19 nm, 90.25 nm and 99.83 nm were found in the mixture with H2O fraction of 50%, 60%, 70%, 80% and 90%, respectively (Fig. 1D). As the increase of H2O fraction, the particle size of the aggregates increased, which also conformed to the characteristics of AIE. Furthermore, the time-resolved emission attenuation behavior of solid TPE-1 was investigated (Fig. S11 and Table S1, respectively), where their attenuation curves can be fitted by a double exponential function, indicating that fluorescence decay involved two relaxation paths. Its weighted average life was 5.55 μs. In addition, TPE-1 showed a higher fluorescence intensity and better stability at the DMF fraction of 20%, so DMF/H2O (v:v = 1:4) was selected in the subsequent experiments.

3.3. The mechanism of the TPE-1 response with pH Frontier molecular orbitals and energy levels of HOMO-1, HOMO, LUMO and LUMO+1 of TPE-1 and TPE-2 were displayed in Table S2. Energy gap is related to the fluorescence emission wavelength, since small energy gap makes electronic transitions more likely to occur, easily resulting in a red-shifted fluorescence emission. In Fig. 3A–D, the electron density in LUMO exhibited the devotion from the delocalized π* orbital on thiophene and phenyl units (electron acceptor), while that of HOMO exhibited main devotion from delocalized π orbital on triphenylamine group (electron donor). Such transition from HOMO to LUMO showed an obvious feature of intra-molecular charge transfer. In solvent, it can form a twisted charge-separate conformation due to the intramolecular rotation, while its excitation state can be non-radioactively decayed; as for the aggregation states, the intramolecular rotation was limited, thus the emission intensity was increased (Fig. S13). Tertiary amine of such molecule is the protonation site, while the attachment of proton to the nitrogen atom can cause the rotation of phenyl group. Fig. 3C and F showed the overlay structure before and after the protonation of TPE-1 and TPE-2, suggesting that the deflection of TPE-1 after protonation was obviously larger than that of TPE-2. According to the restriction of intramolecular motion (RIM) mechanism of AIE [43], the fluorescence activity of TPE-1 was more sensitive to the pH of solvent, which was also consistent with the experimental results. Therefore, the possible mechanism was written as Scheme 2.

3.2. Sensitive fluorescence detection of pH The fluorescence spectra of TPE-1 in DMF/H2O (1/4, v/v) were tested at different pH values. In Fig. 2A, the fluorescence intensity at 446 nm of TPE-1 gradually changed with the increase in pH values, but the change in fluorescence intensity at 593 nm was not very obvious. Therefore, we chose the fluorescence change at 446 nm as the detection wavelength. In Fig. 2B, TPE-1 exhibited weaker fluorescence intensity at 446 nm when the pH value was less than 3.5. As the pH increased, the degree of protonation in TPE-1 decreased gradually, causing the increase of aggregation in the solution, while the fluorescence intensity didn’t increase after the pH was over 7. Therefore, it indicated that TPE1 was pH sensitive under acidic conditions and can be used to detect pH changes. Similarly, the fluorescence spectra of TPE-2 under different pH values were measured under the same conditions (Fig. S12), where the fluorescence intensity of TPE-2 was stronger since it had higher conjugation degree due to the addition of thiophene groups. However, compared with TPE-1, TPE-2 had a lower quantum yield (ФF = 40.56%) and a shorter lifetime (< τ > = 3.25 μs, Table S1). In

3.4. Detection of organophosphorus pesticides (OPs) Considering the hydrolysis product of ATCh can cause the pH change and thus influence the fluorescence of TPE-1, the AChE detection was conducted. As a hydrolase in the nervous system, AChE terminates the transmission of nerve impulses mainly by hydrolyzing neurotransmitter acetylcholine. Since TPE-1 is responsive to the 159

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Fig. 2. (A) Fluorescence spectra of 10 μM TPE-1 at different pH values from 3.20 to 7.73 in DMF/H2O (1/4, v/v). (B) Fluorescence intensity at 446 nm and 593 nm of TPE-1 with different pH values. λex = 365 nm.

were studied. In Fig. S14A, as the concentration of TPE-1 increased, the normalized intensity of the difference in fluorescence intensity before and after the addition of the pesticide gradually increased, reached the highest at 0.1 mM and did not change after 0.1 mM, so 0.1 mM was chosen as the concentration of TPE-1. In Fig. S14B, a longer incubation time can enhance the sensitivity, but it maintained at a relatively stable value after 15 min, indicating that the reaction was supersaturated, so the response time was chosen as 15 min. In Fig. S14C, a low pH can cause the denaturation of enzymes, thus the sensitivity was low. As the pH increased to 7.2, the sensitivity gradually increased. However, as the pH was further increased, the sensitivity decreased, probably due to the decomposition of OPs under alkaline conditions. Therefore, pH 7.2 was chosen for OPs detection. Under the proper conditions, different concentrations of dimethoate were incubated with AChE for 15 min and then added to the AIE-ATCh system. Besides, all measurements were proceeded in DMF/H2O (1:4, v:v, pH = 7.2). The concentrations of AChE and ATCh were 2.5 mU and 0.3 M, respectively. As the concentration of dimethoate increased, the fluorescence intensity at 446 nm increased significantly (Fig. 5A and B). To be specific, the fluorescence intensity of the AIE-AChE-ATCh system

variation of pH values under the acidic condition, a mixed solvent of pH 7.2 buffer and DMF (1/4, v/v) was introduced in the reaction. In Fig. 4A, as the amount of ATCh increased, the fluorescence intensity gradually decreased. To be specific, after the addition of 120 μL ATCh, the fluorescence intensity decreased about 25%. In Fig. 4B, with the increase of AChE concentration, the fluorescence intensity of TPE-1 decreased rapidly and reached equilibrium until about 2.5 mU. The acetic acid produced by the hydrolysis of ATCh by AChE can lower the pH of the reaction system, so the protons can attach to the nitrogen atom on the tertiary amine and its fluorescence intensity can be reduced, which was attributed to the decrease in the aggregation and the increase in the solubility by the increased intermolecular repulsion between protonated TPE-1 molecules [41]. The principle of the OPs detection was shown in Fig. 4C. As we know that OPs can inhibit the activity of AChE irreversibly, leading to the decrease amount of acetic acid, thus the protonation of TPE-1 was lowered, causing the fluorescent signal to recover accordingly. Here, dimethoate was selected as a representative of OPs to inhibit AChE activity. In order to obtain the best detection performance, influnce factors such as TPE-1 concentration, incubation time and initial pH

Fig. 3. (A) The HOMO, (B) the LUMO, and (C) overlays of protonized and unprotonized structures of TPE-1 (green: before protonization, yellow: after protonization); (D) The HOMO, (E) the LUMO, and (F) overlays of protonized and unprotonized structures of TPE-2 (pink: after protonization, white: before protonization) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). 160

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Scheme 2. Proposed pH sensing mechanism using TPE-1. Fig. 4. (A) The relationship of fluorescence intensity at 446 nm of 20 μM TPE-1 with different volumes of ATCh (a) and different concentrations of ATCh (b) in DMF/H2O (1:4, v:v, pH = 7.2), λex = 365 nm. (B) The relationship of fluorescence intensity at 446 nm with various concentrations of AChE. (C) The principle of OPs detection.

difference of fluorescence intensity between the dimethoate in the presence or absence of the AIE-AChE-ATCh system, and ΔI was that the difference of fluorescence intensity when different types of pesticides were in or not in the AIE-AChE-ATCh system. The results were not surprising, as the detection mechanism was based on the fact that OPs had an inhibitory effect on acetylcholinesterase activity, while other pesticides did not. Therefore, the AIE-AChE-ATCh system was successfully used to detect OPs with high selectivity by utilizing the fluorescence property of TPE-1 in different pH solutions. In order to evaluate the practical application of this method, the OPs in the lake water was detected by the standard addition method. As shown in Table 1, the recovery rate of the method was between 98% and 112%. Such considerable accuracy indicated that it can be used to detect OPs in environmental samples.

increased by more than 20% in the range of 0–22.5 mg/L of the OPs. The reason was that the inhibition of AChE by OPs can hinder the enzymatic reaction and decrease the production of acetic acid; while the protonation of TPE-1 was impaired, resulting in the recovery of fluorescence intensity. As can be seen from Fig. 5C, during the range from 0.009 to 22.5 mg/L, the fluorescence intensity had a good linear relationship with the logarithm of dimethoate concentrations (R2 = 0.987) as I = 101.68( ± 4.12)lgC + 1776.84( ± 5.92)(mg L−1), while the detection limit was 0.008 mg/L. The results were comparable or better than earlier work (Table S3), suggesting its sensitivity was acceptable. Specificity is also important in the detection of practical samples. In order to investigate the selectivity, the response of some pesticides (other types of high-efficiency pesticides) including emamectin benzoate (biological insecticides), spirotetramat (quaternary acid ester pesticide), hexythiazox (organochlorine pesticides), tebuconazole (triazole pesticides) and fenpyroximate (pyrazole pesticides) were explored at the same concentration of 5 mg/L. The structural formula of these pesticides can be seen from Fig. S15. In Fig. 5D, the fluorescence variation of such interfering substances was less than 10% of that of the dimethoate, therefore none of such typical pesticides produced significant response to the AIE-AChE-ATCh system. Among it, ΔI0 was the

4. Conclusion Herein, we designed a convenient and sensitive methodology to detect OPs. Basing on the irreversible inhibition ability of OPs on AChE activity, the hydrolysis of ATCh was inhibited to produce acetic acid, which weakened the protonation of TPE-1, thus a signal-on fluorescence detection of OPs was established. Such method can detect OPs in 161

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Fig. 5. (A) Fluorescence spectra of the AIEAChE-ATCh solution with different dimethoate concentrations (from a to j: 0, 0.009, 0.018, 0.09, 0.45, 1.12, 2.25, 4.5, 11.2, 22.5 mg/L), λex = 365 nm. (B) Plots of fluorescence intensity at 446 nm with various concentrations of dimethoate (N = 3). (C) The linear relationship of fluorescence intensity with the logarithm of dimethoate concentrations. (D) The selectivity of this biosensor for OPs detection.

Table 1 Recoveries of the dimethoate detection in lake water by the standard addition. Spiking value (mg/L)

Found (mg/L)

Recovery (%)

0.05 1 5 10

0.056 1.11 4.90 10.11

112 111 98 101.1

2823–2829. [3] X. Yan, H. Li, X. Wang, X. Su, A novel fluorescence probing strategy for the determination of parathion-methyl, Talanta 131 (2015) 88–94. [4] X. Lu, L. Tao, D. Song, Y. Li, F. Gao, Bimetallic Pd@Au nanorods based ultrasensitive acetylcholinesterase biosensor for determination of organophosphate pesticides, Sens. Actuators B-Chem. 255 (2018) 2575–2581. [5] M. Furlong, A. Herring, J. Buckley, B. Goldman, J. Daniels, L. Engel, M. Wolff, J. Chen, J. Wetmur, D. Barr, S. Engel, Prenatal exposure to organophosphorus pesticides and childhood neurodevelopmental phenotypes, Environ. Res. 158 (2017) 737–747. [6] X. Meng, C. Schultz, C. Cui, X. Li, H. Yu, On-site chip-based colorimetric quantitation of organophosphorus pesticides using an office scanner, Sens. Actuators BChem. 215 (2015) 577–583. [7] H. Zhao, X. Ji, B. Wang, N. Wang, X. Li, R. Ni, J. Ren, An ultra-sensitive acetylcholinesterase biosensor based on reduced graphene oxide-Au nanoparticles-βcyclodextrin/Prussian bluechitosan nanocomposites for organophosphorus pesticides detection, Biosens. Bioelectron. 65 (2015) 23–30. [8] C. Wang, A. Periasamy, H. Chang, Photoluminescent C-dots@RGO probe for sensitive and selective detection of acetylcholine, Anal. Chem. 85 (2013) 3263–3270. [9] T. Zhou, X. Xiao, G. Li, Microwave accelerated selective soxhlet extraction for the determination of organophosphorus and carbamate pesticides in ginseng with gas chromatography/mass spectrometry, Anal. Chem. 84 (2012) 5816–5822. [10] D. Huo, Q. Li, Y. Zhang, C. Hou, Y. Lei, A highly efficient organophosphorus pesticides sensor based on CuO nanowires-SWCNTs hybrid nanocomposite, Sens. Actuators B-Chem. 199 (2014) 410–417. [11] T. Hou, L. Zhang, X. Sun, F. Li, Biphasic photoelectrochemical sensing strategy based on in situ formation of CdS quantum dots for highly sensitive detection of acetylcholinesterase activity and inhibition, Biosens. Bioelectron. 75 (2016) 359–364. [12] K. Seebunrueng, Y. Santaladchaiyakit, S. Srijaranai, Vortex-assisted low density solvent liquid–liquid microextraction and salt-induced demulsification coupled to high performance liquid chromatography for the determination of five organophosphorus pesticide residues in fruits, Talanta 132 (2015) 769–774. [13] Q. Long, H. Li, Y. Zhang, S. Yao, Upconversion nanoparticle-based fluorescence resonance energy transfer assay for organophosphorus pesticides, Biosens. Bioelectron. 68 (2015) 168–174. [14] S. Liao, W. Han, H. Ding, H. Tan, S. Yang, Z. Wu, G. Shen, R. Yu, Modulated dye retention for the signal-on fluorometric determination of acetylcholinesterase inhibitor, Anal. Chem. 85 (2013) 4968–4973. [15] C. Niu, Q. Liu, Z. Shang, L. Zhao, J. Ouyang, Dual-emission fluorescent sensor based on AIE organic nanoparticles and Au nanoclusters for the detection of mercury and melamine, Nanoscale 7 (2015) 8457–8465. [16] X. Dou, X. Chu, W. Kong, J. Luo, M. Yang, A gold-based nanobeacon probe for fluorescence sensing of organophosphorus pesticides, Anal. Chim. Acta 891 (2015) 291–297. [17] S. Xiao, Z. Chu, X. Zhao, Z. Zhang, Y. Liu, Off-on-off detection of the activity of acetylcholine esterase and its inhibitors using MoOx quantum dots as a photoluminescent probe, Microchim. Acta 184 (2017) 4853–4860.

the range from 0.009 mg/L to 22.5 mg/L with the detection limit of 0.008 mg/L. Therefore, the present works not only provide a new method for designing pH-sensitive fluorescence probes, but also a new strategy for OPs detection in environment and agriculture. Acknowledgements This work was supported by the National Natural Scientific Foundation of China (21874048, 21705051), the Scientific Foundation of Guangdong Province (2017A030313077), the Program for the Top Young Innovative Talents of Guangdong Province (2016TQ03N305), the Science and Technology Planning Project of Guangzhou City (201607010349), the Science and Technology Planning Project of Shenzhen City (JCYJ20180307164055935), and the Foundation for High-level Talents in South China Agricultural University. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2019.04.123. References [1] X. Liu, M. Song, T. Hou, F. Li, Label-free homogeneous electroanalytical platform for pesticide detection based on acetylcholinesterase-mediated DNA conformational switch integrated with rolling circle amplification, ACS Sens. 2 (2017) 562–568. [2] H. Chen, H. Zhang, R. Yuan, S. Chen, Novel double-potential electrochemiluminescence ratiometric strategy in enzyme-based inhibition biosensing for sensitive detection of organophosphorus pesticides, Anal. Chem. 89 (2017)

162

Sensors & Actuators: B. Chemical 292 (2019) 156–163

Y. Cai, et al.

[36] J. Chang, H. Li, T. Hou, F. Li, Paper-based fluorescent sensor for rapid naked-eye detection of acetylcholinesterase activity and organophosphorus pesticides with high sensitivity and selectivity, Biosens. Bioelectron. 86 (2016) 971–977. [37] G. Li, K. Jiang, Y. Li, S. Li, L. Yang, Efficient structural modification of triphenylamine-based organic dyes for dye-sensitized solar cells, J. Phys. Chem. C 112 (2008) 11591–11599. [38] A. Grüneis, G. Booth, M. Marsman, J. Spencer, A. Alavi, G. Kresse, Natural orbitals for wave function based correlated calculations using a plane wave basis set, J. Chem. Theory Comput. 7 (2011) 2780–2785. [39] H. Zhou, J. Mei, Y. Chen, C. Chen, W. Chen, Z. Zhang, J. Su, P. Chou, H. Tian, Phenazine-based ratiometric Hg2+ probes with well-resolved dual emissions: a new sensing mechanism by vibration-induced emission (VIE), Small 12 (2016) 6542–6546. [40] S. Fateminia, Z. Wang, C. Goh, P. Manghnani, W. Wu, D. Mao, L. Ng, Z. Zhao, B. Tang, B. Liu, Nanocrystallization: a unique approach to yield bright organic nanocrystals for biological applications, Adv. Mater. 29 (2017) 1604100. [41] L. Viglianti, N. Leung, N. Xie, X. Gu, H. Sung, Q. Miao, L. Williams, E. Licandro, B. Tang, Aggregation-induced emission: mechanistic study of the clusteroluminescence of tetrathienylethene, Chem. Sci. 8 (2017) 2629–2639. [42] H. Yan, X. Meng, B. Li, S. Ge, Y. Lu, Design, synthesis and aggregation induced emission properties of two bichromophores with a triphenylamine-coumarin dyad structure, Dyes Pigm. 146 (2017) 479–490. [43] Y. Cai, C. Gui, K. Samedov, H. Su, X. Gu, S. Li, W. Luo, H. Sung, J. Lam, R. Kwok, L. Williams, A. Qin, B. Tang, An acidic pH independent piperazine-TPE AIEgen as a unique bioprobe for lysosome tracing, Chem. Sci. 8 (2017) 7593–7603.

[18] J. Hou, Z. Tian, H. Xie, Q. Tian, S. Ai, A fluorescence resonance energy transfer sensor based on quaternized carbon dots and Ellman’s test for ultrasensitive detection of dichlorvos, Sens. Actuators B-Chem. 232 (2016) 477–483. [19] M. Wang, K. Su, J. Cao, Y. She, A. El-Aty, A. Hacımüftüoğlu, J. Wang, M. Yan, S. Hong, S. Lao, Y. Wang, “Off-On” non-enzymatic sensor for malathion detection based oon fluorescence resonance energy transfer between β-cyclodextrin@Ag and fluorescent probe, Talanta 192 (2019) 295–300. [20] D. Kumar, S. Alex, R. Kumar, N. Chandrasekaran, A. Mukherjee, Acetylcholinesterase inhibition-based ultrasensitive fluorometric detection of malathion using unmodified silver nanoparticles, Colloid Surf. A: Physicochem. Eng. 485 (2015) 111–117. [21] A. Mukhametshina, S. Fedorenko, I. Zueva, K. Petrov, P. Masson, I. Nizameev, A. Mustafina, O. Sinyashin, Luminescent silica nanoparticles for sensing acetylcholinesterase-catalyzed hydrolysis of acetylcholine, Biosens. Bioelectron. 77 (2016) 871–878. [22] M. Sun, M. Su, H. Sun, Spectroscopic investigation on the interaction characteristics and inhibitory activities between baicalin and acetylcholinesterase, Med. Chem. Res. 27 (2018) 1589–1598. [23] S. Wu, D. Li, Z. Gao, J. Wang, Controlled etching of gold nanorods by the Au(III)CTAB complex, and its application to semi-quantitative visual determination of organophosphorus pesticides, Microchim. Acta 184 (2017) 4383–4391. [24] D. Ding, K. Li, B. Liu, B. Tang, Bioprobes based on AIE fluorogens, Acc. Chem. Res. 46 (2013) 2441–2453. [25] H. Ma, C. He, X. Li, O. Ablikim, S. Zhang, M. Zhang, A fluorescent probe for TNP detection in aqueous solution based on joint properties of intramolecular charge transfer and aggregation-induced enhanced emission, Sens. Actuators B-Chem. 230 (2016) 746–452. [26] Z. Peng, X. Feng, B. Tong, D. Chen, J. Shi, J. Zhi, Y. Dong, The selective detection of chloroform using an organic molecule with aggregation-induced emission properties in the solid state as a fluorescent sensor, Sens. Actuators B-Chem. 232 (2016) 264–268. [27] H. Qian, M. Cousins, E. Horak, A. Wakefield, M. Liptak, I. Aprahamian, Suppression of Kasha’s rule as a mechanism for fluorescent molecular rotors and aggregationinduced emission, Nat. Chem. 9 (2017) 83–87. [28] H. Li, J. Chang, P. Gai, Li F, Label-free and ultrasensitive biomolecule detection based on aggregation induced emission fluorogen via target-triggered hemin/Gquadruplex- catalyzed oxidation reaction, ACS Appl. Mater. Interfaces 10 (2018) 4561–4568. [29] H. Li, C. Wang, T. Hou, F. Li, Amphiphile-mediated ultrasmall aggregation induced emission dots for ultrasensitive fluorescence biosensing, Anal. Chem. 89 (2017) 9100–9107. [30] S. Liu, Y. Cheng, H. Zhang, Z. Qiu, R. Kwok, J. Lam, B. Tang, In situ monitoring of RAFT polymerization by tetraphenylethylene-containing agents with aggregationinduced emission characteristics, Angew. Chem. Int. Ed. 57 (2018) 6274–6278. [31] X. Han, B. Zhang, J. Chen, S. Liu, C. Tan, H. Liu, M. Lang, Y. Tan, X. Liu, J. Yin, Modulating aggregation-induced emission via a non-conjugated linkage of fluorophores to tetraphenylethenes, J. Mater. Chem. B 5 (2017) 5096–5100. [32] L. Wang, L. Yang, D. Cao, Probes based on diketopyrrolopyrrole and anthracenone conjugates with aggregation-induced emission characteristics for pH and BSA sensing, Sens. Actuators B-Chem. 221 (2015) 155–166. [33] Y. Wu, S. Huang, F. Zeng, J. Wang, C. Yu, J. Huang, H. Xie, S. Wu, A ratiometric fluorescent system for carboxylesterase detection with AIE dots as FRET donors, Chem. Commun. 51 (2015) 12791–12794. [34] M. Wang, X. Gu, G. Zhang, D. Zhang, D. Zhu, Convenient and continuous fluorometric assay method for acetylcholinesterase and inhibitor screening based on the aggregation-induced emission, Anal. Chem. 81 (2009) 4444–4449. [35] M. Yang, H. Zhou, Y. Zhang, Z. Hu, N. Niu, C. Yu, Controlled synthesis of polydopamine: a new strategy for highlysensitive fluorescence turn-on detection of acetylcholinesterase activity, Microchim. Acta 185 (2018) 132.

Yue Cai received his B.S. degree from South China Agricultural University in 2018. He is currently doing his M.S. work in College of Material and Energy, South China Agricultural University, China. His research interests mainly focus on the application of fluorescent probes in environmental analysis. Jingkun Fang received his B.S. degree in 2005 and Ph.D. in chemistry in 2010 from Hunan University. He is currently an associate professor at the Department of Chemistry, School of Chemical Engineering, Nanjing University of Science and Technology, Nanjing, China. His research interests concern the organic synthesis and functional molecules. Bingfeng Wang received his B.S. degree in 2001 and master degree in computer chemistry in 2004 from South China Normal University. He is currently a teacher in the Department of Applied Chemistry, College of Materials and Energy, South China Agricultural University, Guangzhou, China. His research interests concern the computer chemistry. Fangshuai Zhang obtained his Ph.D. degree from Sun Yat-sen University in 2018. He is currently a postdoctoral research fellow at the Department of Chemistry, School of Chemistry, Sun Yat-sen University. His primary research interests concern organic inorganic hybrid for optical and electric application. Guang Shao obtained his bachelor and master degree from Hunan University in 2000 and 2003, respectively, and his Ph.D. degree from Okayama University of Science in 2007. He is currently an associate professor in School of Chemistry, Sun Yat-sen University. His primary research interests concern the synthesis of organic semiconductor materials and their applications. Yingju Liu received her B.S. degree in 2000 and Ph.D. in chemistry in 2005 from Hunan University. She is currently a full-time professor in the Department of Applied Chemistry, College of Materials and Energy, South China Agricultural University, Guangzhou, China. Her primary research interests concern the biosensor and nanomaterials.

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