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Fluorescent peptide probes for organophosphorus pesticides detection
Journal of Hazardous Materials 389 (2020) 122074
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Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Fluorescent peptide probes for organophosphorus pesticides detection a
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Jianying Wang , Jiaying Zhang , Jing Wang , Guozhen Fang , Jifeng Liu *, Shuo Wang a b
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State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology, Tianjin, 300457, PR China Research Center of Food Science and Human Health, School of Medicine, Nankai University, Tianjin, 300071, PR China
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Editor: R. Teresa
Extensive use of organophosphorus pesticides (OPs) in crop protection has aroused worldwidely great concern about safety and the detection of OPs is of great significance to food safety and human health. In this work, peptides attached with tetraphenylethylene (TPE) molecule were synthesized to from an aggregation-induced emission fluorescent probe (TPE-Peptide) for the determination of OPs. The working mechanism was as follows: in presence of OPs, OPs would react with active site serine in the peptide sequence via covalent bond and adducts were formed between OPs and the peptides; once formed, the adducts accelerated the aggregation of peptides, thus inducing strong emission of TPE-Peptide probe. So the adducts formation and the enhanced emission of the TPE-Peptide probe were the key factors for the OPs' sensing. Herein, the adducts formed between OPs and TPE-Peptide probe, the aggregated peptide fibrils were characterized by fluorescence, mass spectrometry, transmission electron microscopy, dynamic light scattering, atomic force microscopy, circular dichroism spectra and confocal fluorescence microscopy etc. This TPE-Peptide probe displayed highly sensitive fluorescence response where OPs' concentrations ranged from 1 to 100 μM with the limit of detection 0.6 μM and also showed selectivity.
Keywords: Fluorescent peptide probe Organophosphorus pesticides Aggression induced emission
1. Introduction Pesticides are crucial substances used in commercial agriculture and allow us to significantly increase crop yields (Pang et al., 2016; Mishra et al., 2017) and among the pesticides organophosphorus pesticides
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(OPs) are the most effective compounds and are widely used in the agriculture at present (Yan et al., 2015; Zhang et al., 2014). However, due to the relatively long half-life and improper use, OPs’ residues cause serious pollution to agricultural products, environment and water system (Zambonin et al., 2004; Bala et al., 2016). Furthermore, OPs are
Corresponding authors at: State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology, Tianjin, 300457, PR China. E-mail addresses: [email protected] (J. Liu), [email protected] (S. Wang).
https://doi.org/10.1016/j.jhazmat.2020.122074 Received 11 November 2019; Received in revised form 3 January 2020; Accepted 10 January 2020 Available online 15 January 2020 0304-3894/ © 2020 Elsevier B.V. All rights reserved.
Journal of Hazardous Materials 389 (2020) 122074
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a neurotoxin and hinder the cholinesterase’s activity even at low concentrations and pose a health hazard to consumers (Kamerlin et al., 2013; Fahimi-Kashani and Hormozi-Nezhad, 2016). Therefore, the monitoring and development of detection techniques of OPs are of great scientific significance to food safety and human health. Up to now, the routine techniques of determining OPs are GC, HPLC methods or chromatography separation coupled with MS detection etc. However, these techniques usually need relatively complicate procedures or pretreatment prior to analysis, and these instruments cannot be used for in situ detection (Niazi and Yazdanipour, 2007). Novel analytical techniques for OPs’ detection with, simple and fast procedure and low cost instruments are in great demand and have been an active field in analytical chemistry and food safety. For example, a number of new sensing technologies have been developed for detection of OPs, such as electrochemical analysis (Zhao et al., 2013; Dong et al., 2015), chemiluminescence (Ouyang et al., 2015; Hou et al., 2016, 2015), ELISA (Wang et al., 2011; Yan et al., 2014) and fluorescence detection with quantum dots used as sensing material, etc. (Yan et al., 2018). The subject of organic fluorophore with aggregation induced emission (AIE) properties have aroused intense interest since its first report in 2001 (Luo et al., 2001). Up to now, thousands of work have been published in the area of synthesis of AIE probes, their applications in biosensing and bioimaging etc. (Liang et al., 2015). Usually, AIE fluorogens (AIEgen) cannot emit light in free molecular states, but they give enhanced emission when they are aggregated or assembled together and this emission mechanism was interpretated as the restricted intermolecular movements (Hong et al., 2011). Compared with the traditional fluorescent probes, AIE has excellent optical stability, higher tolerance to photobleaching and higher signal reliability (Mei et al., 2014, 2015; Ding et al., 2013). Therefore, in this work, AIE mechanism and the AIE probes were investigated to help design new biosensor techniques for OPs’ detection. The toxicity of the OPs was originated from their ability to form a covalent bond between the phosphate ester group of OPs and active site serine in acetylcholinesterase (AChE) (Sun and Lynn, 2007). Therefore the accumulated acetylcholine in nervous system lead to fatal consequences (Liu et al., 2012). Inspired by this reaction mechanism, herein, an AIE molecule conjugated peptide probe for selective detection of OPs was developed. This probe was comprised of three components: i) an AIEgen TPE reporter, which given emission when molecules were aggregated; ii) a peptide sequence, LHLHLRL, which is easily aggregated via alternating hydrophilic and hydrophobic interactions. In this work, TPE reporter was bound to LHLHLRL sequence by the reaction of aldehyde amine chemistry at the amine site of arginine; iii) a catalytic triad sequence containing serine acting as binding site toward OPs. In presence of OPs, the serine site within the peptide sequence react with OPs by attaching a phosphate ester group via nucleophilic route to form covalent bond (Sun and Lynn, 2007). So, adducts formed between OPs and peptide probe enhances the hydrophobicity of the probe, which would be aggregated into peptide fibrils more easily and induce enhanced emission of TPE-Peptide. This TPE-Peptide as an OPs fluorescent probe had the advantage that the probe’s properties can be modified via adjusting the amino acids’ sequence and the bound sites of TPE reporter. Recently peptide based biomaterials have been applied into analytical chemistry, chemical biology, etc. (Acar et al., 2017), such as biosensors for pesticides (Neupane et al., 2017; Neupane et al., 2016), drug delivery (Wu et al., 2015), metal ions sensing (He et al., 2018) and our previous reports on biomimic materials (He et al., 2017a, b).
Table 1 TPE-Peptide probes used in this Work. Probe(peptide-TPE)
Amino Acid Sequence
Basic peptide P1 AC-SLHLHLRL-CONH2 Validation of active site serine P2 AC-LHLHLRL-CONH2 Validation of the LHLHLRL sequence P3 AC-SLRL-CONH2 Addition of catalytic triad P4 AC-HSHLHLHLRL-CONH2 P5 AC-SHHLHLHLRL-CONH2 P6 AC-SHDLHLHLRL-CONH2 P7 AC-SHELHLHLRL-CONH2 P8 AC-SKDLHLHLRL-CONH2 P9 AC-SKELHLHLRL-CONH2 P10 AC-SEDLHLHLRL-CONH2 P11 AC-SSKLHLHLRL-CONH2
MW(g/mol)
1406.14 1319.06 475.59 1680.36 1680.47 1658.62 1672.45 1649.28 1663.36 1650.39 1621.87
indexed they have been purified by HPLC and characterized by MALDITOF-MS and received under lyophilized conditions. An AIEgen TPE with an aldehyde group (TPE-CHO) was synthetized by our group previously (He et al., 2018) following the procedures reported by Nikhil R. Jana (Pradhan et al., 2015a) and was described briefly in Scheme S1. The purity, structure and the characterization of TPE-CHO have been described in our previous work (He et al., 2018). Pesticides including methyl paraoxon, ethyl paraoxon, carbaryl, methomyl were purchased from Sigma-Aldrich (USA). Methyl parathion was purchased from o2si (USA). Ultrapure water prepared by a Milli-Q system (18.2 MΩ, Millipore) was used in this work. The fluorescence spectrum of the samples were obtained from a F2500 fluorescence spectroscopy instrument (Hitachi, Japan). Solutions to be tested were dropped onto a glass slide, which was imaged with an ultra-high resolution laser confocal fluorescence microscope under UV excitation (CFM, Zeiss 880). Dynamic light scattering (DLS) were obtained by a BT-90 nano laser particle size analyzer. Transmission electron microscopy (TEM) images were obtained from a JEOL JEM2100 (Japan) electron microscopy with the acceleration voltage was set at 200 kV. Atomic force microscopy (AFM) images were obtained with ScanAsyst Mode (PeakForce Tapping) by a Bruker MultiMode 8 atomic force microscope with sharpened Si3N4 probes (radius of curvature about 2 nm, Bruker). Circular dichroism spectra (CD) were obtained by a enzyme kinetics rapid analysis system (Chirascan, Britain). 2.2. Synthesis of peptide-functionalized TPE (TPE-Peptide) Compound TPE-CHO was bound to the arginine site of the peptide sequence LHLHLRL by the reaction of aldehyde amine according to the procedures reported previously (Pradhan et al., 2015b) and some modifications were made. TPE-CHO (3.2 mg) was dissolved in ethanol (1200 μL). 8.0 mg of peptide lyophilized powder was dissolved in ethanol-water (1200 μL, 1:1 v/v). TPE-CHO and peptide were mixed together, and 400 μL mixed solution of triethylamine-ethanol (1:1 v/v) was added into the mixed TPE-CHO and peptide solutions and the mixture was kept at 4 ℃ for 30 min under stirring, and then 200 μL of NaBH4 in ethanol (18.5 mg/mL) was added. The reaction was allowed to be continued overnight at 4 ℃ and the product (TPE-Peptide adducts) was separated and confirmed by mass spectroscopy. Mass spectral measurements of the obtained TPE-Peptides adducts were conducted using a Bruker ultraflextreme MALDI-Mass spectrometer equipped with a nitrogen laser (337 nm) and α-cyano-4-hydroxycinnamic acid (CHCA) was used as matrix.
2. Experimental 2.1. Reagents and apparatus
2.3. Reaction of OPs and TPE-Peptide probe All sequences of peptides (98 %) (sequences as listed in Table 1) were purchased from Sangon Biotech Co. Ltd. (Shanghai, China). As
TPE-Peptide probe (50 μM) and OPs (50 μM) were mixed together in 2
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Scheme 1. Schematic diagram of working principle.
600 μL of phosphate buffer (PBS, 1 mM, pH 7.0) and incubated at 37 ℃ for 15 min and then the fluorescence intensity was measured. The fluorescence spectrum of the samples were recorded by exciting at 365 nm. The adducts formed after reaction of OPs and the active probes (P4, P8, P9 and P11) were verified by using MALDI-TOF-MS. Solutions to be analyzed were mixed with the CHCA matrix (1:1, v/v), and about 2 μL of the sample solution was placed onto the sample array plate and dried under ambient condition. Mass spectrometry was obtained in a positive ion nonlinear mode with an accelerating voltage of 25 kV. In addition, TEM, AFM, SEM, CFM, DLS and CD were conducted to observe the TPEPeptide probe before and after reacted with OPs.
methyl parathion) in acetone was injected manually in splitless mode into a GC–MS instrument (Scion TQ GC–MS, Bruker Inc.) for measurement. The flow rate of helium carrier gas was about 1 mL/min. The initial column temperature was set at 80 ℃ for 2 min, and then rose gradually to 280 ℃ at an increment of 10 ℃/min. The retention time of methyl paraoxon, methyl parathion and ethyl paraoxon were 18.130, 18.548 and 18.935 min, respectively.
3. Results and discussion 3.1. Design of TPE-Peptides probes
2.4. Binding stoichiometry of TPE-Peptide probe and OPs
AChE is a serine hydrolase that catalyzes the dissocation of acetylcholine in cholinergic synapses in biosystems (Cai et al., 2019). The serine sites within the catalytic traid is responsible for binding with organophosphorus anhydride and the resulted OP-AChE conjugate is regarded to irreversibly deactivate AChE (Millard et al., 1999). So this inhibitation process start form phosphorylation of the serine active site and followed with post-inhibitory reactions or aging, which lead to total AChE inhibition (Millard et al., 1999).The design of peptide probe for OPs’ sensing in this work was inspired by this phosphorylation mechanism of serine. After reaction of OPs with the peptide containing serine, the phosphorylated adducts has enhanced hydrophobicity and aggregated more easily than the original peptides, and then TPE molecule attached onto the peptide probe would give an enhanced emission. So this TPE-peptide probe would be applied into OPs’ detection. Scheme 1 showed the working mechanism of this TPE-peptide probe. The amino acids sequences of TPE-Peptide probe for the OPs’ detection are shown in Table 1. The probe consists of TPE as an AIE reporter, catalytic triads containing serine as OPs binding or phosphorylation site, and a peptide sequence of LHLHLRL that can be selfassembled or aggregated via alternating hydrophobic and hydrophilic interactions (Rufo et al., 2014). The catalytic triad generally refers to three amino acid residues that act simultaneously in the center of the active site of a hydrolase (Qiu et al., 2019). During hydrolysis reaction, the deprotonated site of the first amino acid (e.g. serine, eCH3OH) nucleophilically attack the OPs molecules and the proton was transferred from residue of second amino acid to the CeOOH group of the third amino acid (e.g. glutamic acid, aspartic acid), thus consisting a “proton-transfer relay” system. P1 contained serine but without whole catalytic triad, P2 contained only LHLHLRL sequence, and P3 consisted
The continuous variation (Job's plot) method was used to investigate the binding stoichiometry of the peptide probe with OPs molecules (Barman et al., 2018) and the variations in fluorescence intensity with various solutions of TPE-Peptide probe and OPs were recorded. The molar concentrations of the TPE-Peptide probe and OPs ([OPs] + [peptide probe] =100 μM) were kept constant but the mole ratios between TPE-Peptide probe and OPs were varied in the range of 0-0.7. 2.5. Determination of the OPs in real samples The real samples were prepared according to the NY/T 761-2008 (China) with some modifications: 25 g of cabbage spoiled by OPs were mixed with 50 mL acetonitrile and then homogenized for 2 min with a high-speed. After filtered the mixture was collected into a 100 mL mixing cylinder with stopper and added with 5−7 g of NaCl and then shaken violently for 1 min. The organic phase was separated from the aqueous phase after the mixture solution was stood for 30 min at room temperature and 15 mL of acetonitrile solution was taken and added into 20 mL centrifuge tube and then it was incubated at 80℃ while high purity N2 gas flow passed slowly through the tube until organic solution was dried. The OPs collected was dissolved with 1 mL of acetone into the centrifuge tube and stored in a refrigerator (−18 ℃). Prior to GC–MS analysis, the OPs solution was filtered using a 0.2 μm filter membrane. 1.0 μL of different concentrations (10, 25, 50, 75, 100 μM) of mixed standard and extracted OPs (methyl paraoxon, ethyl paraoxon and 3
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serine but without LHLHLRL sequence. The sequences of P4 to P11 consisted of LHLHLRL and HSH, SHH, SHD, SHE, SKD, SKE, SED and SSK catalytic triads, respectively. The TPE was attached to peptide via aldehyde groups of TPE−CHO and the side chain amino of arginine in the LHLHLRL sequence. The probes P4, P8, P9 and P11 were found to give higher emission after reacted with OPs and these four probes were investigated in the following work.
℃, while for P9, the optimized temperature was 25 ℃. The reaction temperature may have effects on the configuration of the active sites or the catalytic triad’s orientation depending on the amino acid sequences. The emission reached plateau after 15 min and reaction time was set at 15 min to achieve a fast detection (Fig. S1D).
3.2. Adducts formation between TPE-peptide and OPs
To better understand the aggregated state of TPE-Peptide probe before and after reaction with OPs, TEM, AFM, SEM, CFM, DLS,CD spectroscopy were conducted. The results of TEM and AFM were shown in Figs. 2 and 3, respectively. TPE-Peptide-OPs adducts for P4, P8, P9, P11 were self-assembled into highly entangled nanofibrous networks. TEM images showed that these probes were in amorphous states (Fig. 2). These amorphous morphologies can also be observed via AFM (Fig. 3). It can be concluded that reactions with OPs can cause the TPE-Peptide probes to be aggregated into nanofibrous network and this aggregation induced enhanced emission of TPE reporter. SEM images were listed in Fig. S4. The TPE-Peptide-OPs adducts for P4, P8, P9, P11 were self-assembled into highly entangled nanofibrous networks and the fiber with several μm size were observed. SEM was in correlation with TEM and AFM results. The secondary structures of TPE-Peptide probes were characterized by CD spectroscopy. As shown in Fig. 4A, P4, P8, P9 and P11 all have negative peaks at 200 nm and positive peaks at 212 nm, which are typical characteristics of random coil structures. As shown in Fig. 4B, the TPE-Peptide-OPs adducts for the probes of P4, P8 and P11 have two positive peaks at 195 nm (positive peak of β-sheet) and 212 nm (positive peak of random coil), respectively, indicating the presence of mixed structures of β-sheet and random coil in TPE-peptide-OPs adducts. P9 has a positive peak at 195 nm and a negative peak at 216 nm, which was a typical β-sheet structure. So TPE-peptide-OPs adducts consisted of more secondary structure than probes P4, P8 and P11. The aggregation of TPE-peptide-OPs adducts were also investigated using DLS measurement. As shown in Fig. S5, the sizes of probe P4, P8, P9 and P11without OPs were 907 ± 83 nm, 1184 ± 105 nm, 975 ± 57 nm and 1284 ± 124 nm, respectively. The sizes were between 0.9 and 1.3 μm. The size values of TPE- peptide probes were slightly different due to the differences in peptide sequences. The sizes of probe P4, P8, P9 and P11with OPs were 2914 ± 151 nm, 3543 ± 212 nm, 3345 ± 178 nm and 3981 ± 243 nm, respectively. The sizes were between 2.9 μm and 4 μm. The sizes of the adducts were larger than the free TPE-Probes (Fig. S5). From these morphology analysis, it can be concluded that the enhanced emission of TPE-Peptide in presence of OPs was mostly resulted from the formation of the aggregated TPE-Peptide-OPs adducts.
3.4. Characterization of the TPE-Peptide probe
For comparison, the emission of TPE-CHO and TPE-Peptide probe in presence or absence of OPs were investigated for the probability of TPEPeptide probe in OPs’ detection. As seen from Fig. S1A, TPE-CHO showed no enhanced emission in presence of OPs. Comparatively, the TPE-peptide probes showed enhanced emission after reacted with OPs. In other words, the fluorescence of TPE-peptide can be turned on in presence of OPs, indicating that the TPE-Peptide probe could be used for OPs’ sensing. The OPs’ detection performances of the 11 probes were demonstrated in Fig. S2. Obviously, the TPE-peptide probes P4, P8, P9, P11 gave higher emission among the 11 probes investigated and P11 gave the highest emission intensity. MALDI-TOF-MS were performed to investigate the adducts formed after reaction of OPs with peptide probes. As shown in Fig. 1, the m/z signals of P4, P8, P9, P11 appeared at 1680.36, 1649.28, 1663.36, 1621.87, respectively. After reacted with OPS, the m/z signals of P4, P8, P9, P11 appeared at 1788.23, 1757.56, 1771.52, 1729.04, respectively. Indicating the molecular weight change of 108 was due the adduction of PO3C2H6-[H] to the peptides. Other probes (P1, P5, P6, P7, P10) have weak ability to bind OPs and weak m/z signals were observed for the adducts formed between OPs and peptide P1, P5, P6, P7, P10, respectively (Fig. S3). From these results, it can be inferred that TPE-Peptide with the higher ability to from adducts with OPs would have higher fluorescence intensity. 3.3. AIE condition optimization The reaction conditions including pH (pH 3, 5, 7, 9, 10), temperature (4, 25, 37, 55, 70 ℃) and time (0, 5, 15, 30, 45, 60 min) of TPEPeptide probe reaction with OPs (TPE-Peptide-OPs) were also investigated (Fig. S1). The probe showed weak fluorescence intensity at lower pH (pH ≤ 5) and this might be due to the electron-withdrawing properties of protonated imidazolyl, amino and phenol groups (Yang et al., 2013; Moyer et al., 2014). The highest signal enhancement was found at pH 7.0. The effects of temperature on TPE-Peptide-OPs’ emission was shown in Fig. S1C. The optimal temperature of probe P4, P8 and P11 was at 37
Fig. 1. MALDI-mass spectrum of active TPE- peptide probes (A, B, C, D) without OPs and with OPs. 4
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Fig. 2. Negatively stained TEM images of the active probes without OPs and with OPs. Scale bar: 200 nm.
Fig. 3. AFM images of the active probes without OPs and with OPs.
aggregation, indicating that the TPE-Peptide probe bound with OPs switched on the enhanced fluorescence.
To further confirm whether the TPE-Peptide probes can turn on enhanced emission after bound with OPs or not, the probe or TPEPeptide-OPs adducts were imaged by CFM (Fig. 5). The merged images in Fig. 5 show that the enhanced emission was obtained only after
Fig. 4. Circular dichroism spectroscopy (CD) measurements of the active probes without (A) and with OPs (B). 5
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Fig. 5. Ultra high resolution fluorescence confocal images of the active probeswithout and with OPs from the same area under bright field (B) and fluorescence (F). The merged image showed that fluorescence was observed from aggregate only, suggesting that the TPE-Peptide binds on OPs to “switch-on” fluorescence.
pH 7.0 for 15 min (Fig. 6). Standard addition method was used in order to study the accuracy of the TPE-Peptide probe technique in detection of OPs in real samples. 25, 50, 75, 100 μM were selected as labeling levels and tested for quantitative analysis of the methods and their recovery was found to be ranged from 85.34%–96.25 % (Table S1). Real samples carried with OPs were detected by TPE-Peptide probe technique and there were 87.65 μM methyl paraoxon, 52.89 μM methyl parathion and 65.01 μM ethyl paraoxon, respectively. These samples were also analyzed by GC–MS to investigate the precision of the developed TPE-Peptide probe method (Fig. 6), and the concentrations read by GC–MS were 86.40, 52.04 and 63.84 μM, respectively (Fig. S7). The little variance (< 2 %) indicated the high precision of this TPEPeptide probe method (Table S2). The detection limit were 0.61 μM (15.07 μg/kg, methyl paraoxon), 0.57 μM (14.07 μg/kg, methyl parathion), 0.60 μM (14.84 μg/kg, ethyl paraoxon), respectively, which were lower than the allowable maximum pesticide residue limits of European Union (0.05 mg/kg). It should be noted that at present, the
3.5. Binding stoichiometry The I/I0 (the emission intensity I, in presence of TPE-Peptide-OPs adducts; I0, in absence of OPs) value of TPE-Peptide probe P11 was studied with the probe concentrations ranged from 0 to 70 μM and I/I0 reached highest value at 50 μM. So the TPE-Peptide probe concentration of 50 μM was used in the detection of OPs. As shown in Fig. S6, the highest emission was observed at a 0.5 mol fraction of OPs, indicating that TPE-Peptide may form a 1:1 adduct with OPs and this result was correlated with the MALDI-TOF-MS results (Fig. 1). 3.6. Real sample analysis Samples of cabbage spiked with different concentrations of OPs were processed according to the NY/T 761–2008 and then detected with fluorescent TPE-Peptide probe method after incubation in 37 ℃ at
Fig. 6. OPs can be quantified according to the fluorescence spectrum and a linear working range was constructed for OPs. Fluorescence standard curve of the actual samples of methyl paraoxon (A), ethyl paraoxon (B), methyl parathion (C) with varying concentration. Fluorescence spectrum of the actual samples of methyl paraoxon (a), ethyl paraoxon (b), methyl parathion (c) with varying concentration. 6
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Declaration of Competing Interest There are no conflicts to declare. Acknowledgments This work was supported by Natural Science Foundation of China (Funding, 21575102) and Tianjin Scientific Program (18ZYPTJC00020) and the Open Funds of the State Key Laboratory of Electroanalytical Chemistry (SKLEAC201911). Dr. Haibo Li, Dr. Min Hong and Prof. Lei Wang (Liaocheng University) are thanked for collaboration. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.jhazmat.2020.122074. Fig. 7. Fluorescence intensity of the active probes (P4, 8, 9 and 11) in the presence of OPs (methyl paraoxon, ethyl paraoxon, methyl parathion) and carbamate pesticides (carbaril, methomyl).
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TPE-Peptide probe method can be used to detect the total amount of OPs, but cannot distinguish the specific types of pesticides. 3.7. Sensing of carbamate pesticides by TPE-Peptide probe method Carbamate pesticides also inhibit AChE’s activity and sensing of carbamate pesticides were also investigated using this TPE-Peptide probe method. The active probes P4, P8, P9 and P11were studied for their emission properties before and after they reacted with carbamate pesticides and the reults were shown in Fig. 7. Compared with the OPs’ sensing, TPE-Peptide probe method was less sensitive to carbaryl and methomyl investigated here. This difference may originate from the less hydrophobicity of the TPE-Peptide-carbamate than TPE-Peptide-OPs adducts because that carbaryl and methomyl have less hydrophobic groups (methyl, ester groups) than OPs (Table S3). 4. Conclusions To summarize, we developed a fluorescent TPE-Peptide probes for OPs’ detection by conjugation of AIE molecule with the R amino acid site of alternating hydrophilic and hydrophobic peptide LHLHLRL component and catalytic triads containing serine. The probe formed adducts with OPs and the induced aggregation caused enhancement of emission of TPE. The properties of these probes to detect OPs were investigated by a series of characterizations, including fluorescence spectroscopy, MALDI-TOF-MS, TEM, SEM, CFM, DLS, and CD. OPs can be quantified within 0.6–100 μM and the accuracy and precision of this fluorescent TPE-Peptide technique was investigated and they could meet the demand for real samples’ analysis. The fluorescent probe has the characteristics of high stability, environmental friendly and easy preparation, therefore this technique may find applications in routine assays of OPs. Authors statement Author contributions Jianying Wang has made substantial contributions to the acquisition, analysis, interpretation of the data for the work and drafting the work. Jiaying Zhang and Jing Wang have made contributions to the acquisition, analysis, interpretation of the data for the work. Guozhen Fang have made contributions to correction of the drafting the work. Jifeng Liu has made substantial contributions to the conception and design the work, revision of the draft and approved the final version to be submitted. Shuo Wang has made substantial contributions to the project administration and correction of the drafting the work. 7
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J. Wang, et al. Neupane, L.N., Hwang, G.W., Lee, K.H., 2017. Tuning of the selectivity of fluorescent peptidyl bioprobe using aggregation induced emission for heavy metal ions by buffering agents in 100% aqueous solutions. Biosens. Bioelectron. 92, 179–185. Neupane, L.N., Oh, E.T., Park, H.J., Lee, K.H., 2016. Selective and sensitive detection of heavy metal ions in 100% aqueous solution and cells with a fluorescence chemosensor based on peptide using aggregation-induced emission. Anal. Chem. 88, 3333–3340. Niazi, A., Yazdanipour, A., 2007. Spectrophotometric simultaneous determination of nitrophenol isomers by orthogonal signal correction and partial least squares. J. Hazard. Mater. 146, 421–427. Ouyang, H., Wang, L., Yang, S., Wang, W., Wang, L., Liu, F., Fu, Z., 2015. Chemiluminescence reaction kinetics-resolved multianalyte immunoassay strategy using a bispecific monoclonal antibody as the unique recognition reagent. Anal. Chem. 87, 2952–2958. Pang, S., Yang, T., He, L., 2016. Review of surface enhanced Raman spectroscopic (SERS) detection of synthetic chemical pesticides. Trends Analyt. Chem. 85, 73–82. Pradhan, N., Jana, D., Ghorai, B.K., Jana, N.R., 2015a. Detection and monitoring of amyloid fibrillation using a fluorescence “switch-on” probe. ACS Appl. Mater. Interfaces 7, 25813–25820. Pradhan, N., Jana, D., Ghorai, B.K., Jana, N.R., 2015b. Detection and monitoring of amyloid fibrillation using a fluorescence “switch-on” probe. ACS Appl. Mater. Interfaces 7, 25813–25820. Qiu, L., Lv, P., Zhao, C., Feng, X., Fang, G., Liu, J., Wang, S., 2019. Electrochemical detection of organophosphorus pesticides based on amino acids conjugated nanoenzyme modified electrodes. Sens. Actuators B Chem. 286, 386–393. Rufo, C.M., Moroz, Y.S., Moroz, O.V., Stohr, J., Smith, T.A., Hu, X., DeGrado, W.F., Korendovych, I.V., 2014. Short peptides self-assemble to produce catalytic amyloids. Nat. Chem. 6, 303–309. Sun, J., Lynn, B.C., 2007. Development of a MALDI-TOF-MS method to identify and quantify butyrylcholinesterase inhibition resulting from exposure to
organophosphate and carbamate pesticides. J. Am. Soc. Mass Spectrom. 18, 698–706. Wang, L., Du, D., Lu, D., Lin, C.T., Smith, J.N., Timchalk, C., Liu, F., Wang, J., Lin, Y., 2011. Enzyme-linked immunosorbent assay for detection of organophosphorylated butyrylcholinesterase: a biomarker of exposure to organophosphate agents. Anal. Chim. Acta 693, 1–6. Wu, J., Chen, A., Qin, M., Huang, R., Zhang, G., Xue, B., Wei, J., Li, Y., Cao, Y., Wang, W., 2015. Hierarchical construction of a mechanically stable peptide-graphene oxide hybrid hydrogel for drug delivery and pulsatile triggered release in vivo. Nanoscale 7, 1655–1660. Yan, X., Li, H., Yan, Y., Su, X., 2015. Selective detection of parathion-methyl based on near-infrared CuInS2 quantum dots. Food Chem. 173, 179–184. Yan, X., Li, H., Yan, Y., Su, X., 2014. Developments in pesticide analysis by multi-analyte immunoassays: a review. Anal. Methods 6, 3543–3554. Yan, X., Song, Y., Zhu, C., Li, H., Du, D., Su, X., Lin, Y., 2018. MnO2 nanosheet-carbon dots sensing platform for sensitive detection of organophosphorus pesticides. Anal. Chem. 90, 2618–2624. Yang, Z., Qin, W., Lam, J.W.Y., Chen, S., Sung, H.H.Y., Williams, I.D., Tang, B.Z., 2013. Fluorescent pH sensor constructed from a heteroatom-containing luminogen with tunable AIE and ICT characteristics. Chem. Sci. 4, 3725–3732. Zambonin, C.G., Quinto, M., De Vietro, N., Palmisano, F., 2004. Solid-phase microextraction-gas chromatography mass spectrometry: a fast and simple screening method for the assessment of organophosphorus pesticides residues in wine and fruit juices. Food Chem. 86, 269–274. Zhang, K., Yu, T., Liu, F., Sun, M., Yu, H., Liu, B., Zhang, Z., Jiang, H., Wang, S., 2014. Selective fluorescence turn-on and ratiometric detection of organophosphate using dual-emitting Mn-doped ZnS nanocrystal probe. Anal. Chem. 86, 11727–11733. Zhao, Y., Zhang, W., Lin, Y., Du, D., 2013. The vital function of Fe3O4@Au nanocomposites for hydrolase biosensor design and its application in detection of methyl parathion. Nanoscale 5, 1121–1126.
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Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Fluorescent peptide probes for organophosphorus pesticides detection a
a
a
a
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Jianying Wang , Jiaying Zhang , Jing Wang , Guozhen Fang , Jifeng Liu *, Shuo Wang a b
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State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology, Tianjin, 300457, PR China Research Center of Food Science and Human Health, School of Medicine, Nankai University, Tianjin, 300071, PR China
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Editor: R. Teresa
Extensive use of organophosphorus pesticides (OPs) in crop protection has aroused worldwidely great concern about safety and the detection of OPs is of great significance to food safety and human health. In this work, peptides attached with tetraphenylethylene (TPE) molecule were synthesized to from an aggregation-induced emission fluorescent probe (TPE-Peptide) for the determination of OPs. The working mechanism was as follows: in presence of OPs, OPs would react with active site serine in the peptide sequence via covalent bond and adducts were formed between OPs and the peptides; once formed, the adducts accelerated the aggregation of peptides, thus inducing strong emission of TPE-Peptide probe. So the adducts formation and the enhanced emission of the TPE-Peptide probe were the key factors for the OPs' sensing. Herein, the adducts formed between OPs and TPE-Peptide probe, the aggregated peptide fibrils were characterized by fluorescence, mass spectrometry, transmission electron microscopy, dynamic light scattering, atomic force microscopy, circular dichroism spectra and confocal fluorescence microscopy etc. This TPE-Peptide probe displayed highly sensitive fluorescence response where OPs' concentrations ranged from 1 to 100 μM with the limit of detection 0.6 μM and also showed selectivity.
Keywords: Fluorescent peptide probe Organophosphorus pesticides Aggression induced emission
1. Introduction Pesticides are crucial substances used in commercial agriculture and allow us to significantly increase crop yields (Pang et al., 2016; Mishra et al., 2017) and among the pesticides organophosphorus pesticides
⁎
(OPs) are the most effective compounds and are widely used in the agriculture at present (Yan et al., 2015; Zhang et al., 2014). However, due to the relatively long half-life and improper use, OPs’ residues cause serious pollution to agricultural products, environment and water system (Zambonin et al., 2004; Bala et al., 2016). Furthermore, OPs are
Corresponding authors at: State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology, Tianjin, 300457, PR China. E-mail addresses: [email protected] (J. Liu), [email protected] (S. Wang).
https://doi.org/10.1016/j.jhazmat.2020.122074 Received 11 November 2019; Received in revised form 3 January 2020; Accepted 10 January 2020 Available online 15 January 2020 0304-3894/ © 2020 Elsevier B.V. All rights reserved.
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a neurotoxin and hinder the cholinesterase’s activity even at low concentrations and pose a health hazard to consumers (Kamerlin et al., 2013; Fahimi-Kashani and Hormozi-Nezhad, 2016). Therefore, the monitoring and development of detection techniques of OPs are of great scientific significance to food safety and human health. Up to now, the routine techniques of determining OPs are GC, HPLC methods or chromatography separation coupled with MS detection etc. However, these techniques usually need relatively complicate procedures or pretreatment prior to analysis, and these instruments cannot be used for in situ detection (Niazi and Yazdanipour, 2007). Novel analytical techniques for OPs’ detection with, simple and fast procedure and low cost instruments are in great demand and have been an active field in analytical chemistry and food safety. For example, a number of new sensing technologies have been developed for detection of OPs, such as electrochemical analysis (Zhao et al., 2013; Dong et al., 2015), chemiluminescence (Ouyang et al., 2015; Hou et al., 2016, 2015), ELISA (Wang et al., 2011; Yan et al., 2014) and fluorescence detection with quantum dots used as sensing material, etc. (Yan et al., 2018). The subject of organic fluorophore with aggregation induced emission (AIE) properties have aroused intense interest since its first report in 2001 (Luo et al., 2001). Up to now, thousands of work have been published in the area of synthesis of AIE probes, their applications in biosensing and bioimaging etc. (Liang et al., 2015). Usually, AIE fluorogens (AIEgen) cannot emit light in free molecular states, but they give enhanced emission when they are aggregated or assembled together and this emission mechanism was interpretated as the restricted intermolecular movements (Hong et al., 2011). Compared with the traditional fluorescent probes, AIE has excellent optical stability, higher tolerance to photobleaching and higher signal reliability (Mei et al., 2014, 2015; Ding et al., 2013). Therefore, in this work, AIE mechanism and the AIE probes were investigated to help design new biosensor techniques for OPs’ detection. The toxicity of the OPs was originated from their ability to form a covalent bond between the phosphate ester group of OPs and active site serine in acetylcholinesterase (AChE) (Sun and Lynn, 2007). Therefore the accumulated acetylcholine in nervous system lead to fatal consequences (Liu et al., 2012). Inspired by this reaction mechanism, herein, an AIE molecule conjugated peptide probe for selective detection of OPs was developed. This probe was comprised of three components: i) an AIEgen TPE reporter, which given emission when molecules were aggregated; ii) a peptide sequence, LHLHLRL, which is easily aggregated via alternating hydrophilic and hydrophobic interactions. In this work, TPE reporter was bound to LHLHLRL sequence by the reaction of aldehyde amine chemistry at the amine site of arginine; iii) a catalytic triad sequence containing serine acting as binding site toward OPs. In presence of OPs, the serine site within the peptide sequence react with OPs by attaching a phosphate ester group via nucleophilic route to form covalent bond (Sun and Lynn, 2007). So, adducts formed between OPs and peptide probe enhances the hydrophobicity of the probe, which would be aggregated into peptide fibrils more easily and induce enhanced emission of TPE-Peptide. This TPE-Peptide as an OPs fluorescent probe had the advantage that the probe’s properties can be modified via adjusting the amino acids’ sequence and the bound sites of TPE reporter. Recently peptide based biomaterials have been applied into analytical chemistry, chemical biology, etc. (Acar et al., 2017), such as biosensors for pesticides (Neupane et al., 2017; Neupane et al., 2016), drug delivery (Wu et al., 2015), metal ions sensing (He et al., 2018) and our previous reports on biomimic materials (He et al., 2017a, b).
Table 1 TPE-Peptide probes used in this Work. Probe(peptide-TPE)
Amino Acid Sequence
Basic peptide P1 AC-SLHLHLRL-CONH2 Validation of active site serine P2 AC-LHLHLRL-CONH2 Validation of the LHLHLRL sequence P3 AC-SLRL-CONH2 Addition of catalytic triad P4 AC-HSHLHLHLRL-CONH2 P5 AC-SHHLHLHLRL-CONH2 P6 AC-SHDLHLHLRL-CONH2 P7 AC-SHELHLHLRL-CONH2 P8 AC-SKDLHLHLRL-CONH2 P9 AC-SKELHLHLRL-CONH2 P10 AC-SEDLHLHLRL-CONH2 P11 AC-SSKLHLHLRL-CONH2
MW(g/mol)
1406.14 1319.06 475.59 1680.36 1680.47 1658.62 1672.45 1649.28 1663.36 1650.39 1621.87
indexed they have been purified by HPLC and characterized by MALDITOF-MS and received under lyophilized conditions. An AIEgen TPE with an aldehyde group (TPE-CHO) was synthetized by our group previously (He et al., 2018) following the procedures reported by Nikhil R. Jana (Pradhan et al., 2015a) and was described briefly in Scheme S1. The purity, structure and the characterization of TPE-CHO have been described in our previous work (He et al., 2018). Pesticides including methyl paraoxon, ethyl paraoxon, carbaryl, methomyl were purchased from Sigma-Aldrich (USA). Methyl parathion was purchased from o2si (USA). Ultrapure water prepared by a Milli-Q system (18.2 MΩ, Millipore) was used in this work. The fluorescence spectrum of the samples were obtained from a F2500 fluorescence spectroscopy instrument (Hitachi, Japan). Solutions to be tested were dropped onto a glass slide, which was imaged with an ultra-high resolution laser confocal fluorescence microscope under UV excitation (CFM, Zeiss 880). Dynamic light scattering (DLS) were obtained by a BT-90 nano laser particle size analyzer. Transmission electron microscopy (TEM) images were obtained from a JEOL JEM2100 (Japan) electron microscopy with the acceleration voltage was set at 200 kV. Atomic force microscopy (AFM) images were obtained with ScanAsyst Mode (PeakForce Tapping) by a Bruker MultiMode 8 atomic force microscope with sharpened Si3N4 probes (radius of curvature about 2 nm, Bruker). Circular dichroism spectra (CD) were obtained by a enzyme kinetics rapid analysis system (Chirascan, Britain). 2.2. Synthesis of peptide-functionalized TPE (TPE-Peptide) Compound TPE-CHO was bound to the arginine site of the peptide sequence LHLHLRL by the reaction of aldehyde amine according to the procedures reported previously (Pradhan et al., 2015b) and some modifications were made. TPE-CHO (3.2 mg) was dissolved in ethanol (1200 μL). 8.0 mg of peptide lyophilized powder was dissolved in ethanol-water (1200 μL, 1:1 v/v). TPE-CHO and peptide were mixed together, and 400 μL mixed solution of triethylamine-ethanol (1:1 v/v) was added into the mixed TPE-CHO and peptide solutions and the mixture was kept at 4 ℃ for 30 min under stirring, and then 200 μL of NaBH4 in ethanol (18.5 mg/mL) was added. The reaction was allowed to be continued overnight at 4 ℃ and the product (TPE-Peptide adducts) was separated and confirmed by mass spectroscopy. Mass spectral measurements of the obtained TPE-Peptides adducts were conducted using a Bruker ultraflextreme MALDI-Mass spectrometer equipped with a nitrogen laser (337 nm) and α-cyano-4-hydroxycinnamic acid (CHCA) was used as matrix.
2. Experimental 2.1. Reagents and apparatus
2.3. Reaction of OPs and TPE-Peptide probe All sequences of peptides (98 %) (sequences as listed in Table 1) were purchased from Sangon Biotech Co. Ltd. (Shanghai, China). As
TPE-Peptide probe (50 μM) and OPs (50 μM) were mixed together in 2
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Scheme 1. Schematic diagram of working principle.
600 μL of phosphate buffer (PBS, 1 mM, pH 7.0) and incubated at 37 ℃ for 15 min and then the fluorescence intensity was measured. The fluorescence spectrum of the samples were recorded by exciting at 365 nm. The adducts formed after reaction of OPs and the active probes (P4, P8, P9 and P11) were verified by using MALDI-TOF-MS. Solutions to be analyzed were mixed with the CHCA matrix (1:1, v/v), and about 2 μL of the sample solution was placed onto the sample array plate and dried under ambient condition. Mass spectrometry was obtained in a positive ion nonlinear mode with an accelerating voltage of 25 kV. In addition, TEM, AFM, SEM, CFM, DLS and CD were conducted to observe the TPEPeptide probe before and after reacted with OPs.
methyl parathion) in acetone was injected manually in splitless mode into a GC–MS instrument (Scion TQ GC–MS, Bruker Inc.) for measurement. The flow rate of helium carrier gas was about 1 mL/min. The initial column temperature was set at 80 ℃ for 2 min, and then rose gradually to 280 ℃ at an increment of 10 ℃/min. The retention time of methyl paraoxon, methyl parathion and ethyl paraoxon were 18.130, 18.548 and 18.935 min, respectively.
3. Results and discussion 3.1. Design of TPE-Peptides probes
2.4. Binding stoichiometry of TPE-Peptide probe and OPs
AChE is a serine hydrolase that catalyzes the dissocation of acetylcholine in cholinergic synapses in biosystems (Cai et al., 2019). The serine sites within the catalytic traid is responsible for binding with organophosphorus anhydride and the resulted OP-AChE conjugate is regarded to irreversibly deactivate AChE (Millard et al., 1999). So this inhibitation process start form phosphorylation of the serine active site and followed with post-inhibitory reactions or aging, which lead to total AChE inhibition (Millard et al., 1999).The design of peptide probe for OPs’ sensing in this work was inspired by this phosphorylation mechanism of serine. After reaction of OPs with the peptide containing serine, the phosphorylated adducts has enhanced hydrophobicity and aggregated more easily than the original peptides, and then TPE molecule attached onto the peptide probe would give an enhanced emission. So this TPE-peptide probe would be applied into OPs’ detection. Scheme 1 showed the working mechanism of this TPE-peptide probe. The amino acids sequences of TPE-Peptide probe for the OPs’ detection are shown in Table 1. The probe consists of TPE as an AIE reporter, catalytic triads containing serine as OPs binding or phosphorylation site, and a peptide sequence of LHLHLRL that can be selfassembled or aggregated via alternating hydrophobic and hydrophilic interactions (Rufo et al., 2014). The catalytic triad generally refers to three amino acid residues that act simultaneously in the center of the active site of a hydrolase (Qiu et al., 2019). During hydrolysis reaction, the deprotonated site of the first amino acid (e.g. serine, eCH3OH) nucleophilically attack the OPs molecules and the proton was transferred from residue of second amino acid to the CeOOH group of the third amino acid (e.g. glutamic acid, aspartic acid), thus consisting a “proton-transfer relay” system. P1 contained serine but without whole catalytic triad, P2 contained only LHLHLRL sequence, and P3 consisted
The continuous variation (Job's plot) method was used to investigate the binding stoichiometry of the peptide probe with OPs molecules (Barman et al., 2018) and the variations in fluorescence intensity with various solutions of TPE-Peptide probe and OPs were recorded. The molar concentrations of the TPE-Peptide probe and OPs ([OPs] + [peptide probe] =100 μM) were kept constant but the mole ratios between TPE-Peptide probe and OPs were varied in the range of 0-0.7. 2.5. Determination of the OPs in real samples The real samples were prepared according to the NY/T 761-2008 (China) with some modifications: 25 g of cabbage spoiled by OPs were mixed with 50 mL acetonitrile and then homogenized for 2 min with a high-speed. After filtered the mixture was collected into a 100 mL mixing cylinder with stopper and added with 5−7 g of NaCl and then shaken violently for 1 min. The organic phase was separated from the aqueous phase after the mixture solution was stood for 30 min at room temperature and 15 mL of acetonitrile solution was taken and added into 20 mL centrifuge tube and then it was incubated at 80℃ while high purity N2 gas flow passed slowly through the tube until organic solution was dried. The OPs collected was dissolved with 1 mL of acetone into the centrifuge tube and stored in a refrigerator (−18 ℃). Prior to GC–MS analysis, the OPs solution was filtered using a 0.2 μm filter membrane. 1.0 μL of different concentrations (10, 25, 50, 75, 100 μM) of mixed standard and extracted OPs (methyl paraoxon, ethyl paraoxon and 3
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serine but without LHLHLRL sequence. The sequences of P4 to P11 consisted of LHLHLRL and HSH, SHH, SHD, SHE, SKD, SKE, SED and SSK catalytic triads, respectively. The TPE was attached to peptide via aldehyde groups of TPE−CHO and the side chain amino of arginine in the LHLHLRL sequence. The probes P4, P8, P9 and P11 were found to give higher emission after reacted with OPs and these four probes were investigated in the following work.
℃, while for P9, the optimized temperature was 25 ℃. The reaction temperature may have effects on the configuration of the active sites or the catalytic triad’s orientation depending on the amino acid sequences. The emission reached plateau after 15 min and reaction time was set at 15 min to achieve a fast detection (Fig. S1D).
3.2. Adducts formation between TPE-peptide and OPs
To better understand the aggregated state of TPE-Peptide probe before and after reaction with OPs, TEM, AFM, SEM, CFM, DLS,CD spectroscopy were conducted. The results of TEM and AFM were shown in Figs. 2 and 3, respectively. TPE-Peptide-OPs adducts for P4, P8, P9, P11 were self-assembled into highly entangled nanofibrous networks. TEM images showed that these probes were in amorphous states (Fig. 2). These amorphous morphologies can also be observed via AFM (Fig. 3). It can be concluded that reactions with OPs can cause the TPE-Peptide probes to be aggregated into nanofibrous network and this aggregation induced enhanced emission of TPE reporter. SEM images were listed in Fig. S4. The TPE-Peptide-OPs adducts for P4, P8, P9, P11 were self-assembled into highly entangled nanofibrous networks and the fiber with several μm size were observed. SEM was in correlation with TEM and AFM results. The secondary structures of TPE-Peptide probes were characterized by CD spectroscopy. As shown in Fig. 4A, P4, P8, P9 and P11 all have negative peaks at 200 nm and positive peaks at 212 nm, which are typical characteristics of random coil structures. As shown in Fig. 4B, the TPE-Peptide-OPs adducts for the probes of P4, P8 and P11 have two positive peaks at 195 nm (positive peak of β-sheet) and 212 nm (positive peak of random coil), respectively, indicating the presence of mixed structures of β-sheet and random coil in TPE-peptide-OPs adducts. P9 has a positive peak at 195 nm and a negative peak at 216 nm, which was a typical β-sheet structure. So TPE-peptide-OPs adducts consisted of more secondary structure than probes P4, P8 and P11. The aggregation of TPE-peptide-OPs adducts were also investigated using DLS measurement. As shown in Fig. S5, the sizes of probe P4, P8, P9 and P11without OPs were 907 ± 83 nm, 1184 ± 105 nm, 975 ± 57 nm and 1284 ± 124 nm, respectively. The sizes were between 0.9 and 1.3 μm. The size values of TPE- peptide probes were slightly different due to the differences in peptide sequences. The sizes of probe P4, P8, P9 and P11with OPs were 2914 ± 151 nm, 3543 ± 212 nm, 3345 ± 178 nm and 3981 ± 243 nm, respectively. The sizes were between 2.9 μm and 4 μm. The sizes of the adducts were larger than the free TPE-Probes (Fig. S5). From these morphology analysis, it can be concluded that the enhanced emission of TPE-Peptide in presence of OPs was mostly resulted from the formation of the aggregated TPE-Peptide-OPs adducts.
3.4. Characterization of the TPE-Peptide probe
For comparison, the emission of TPE-CHO and TPE-Peptide probe in presence or absence of OPs were investigated for the probability of TPEPeptide probe in OPs’ detection. As seen from Fig. S1A, TPE-CHO showed no enhanced emission in presence of OPs. Comparatively, the TPE-peptide probes showed enhanced emission after reacted with OPs. In other words, the fluorescence of TPE-peptide can be turned on in presence of OPs, indicating that the TPE-Peptide probe could be used for OPs’ sensing. The OPs’ detection performances of the 11 probes were demonstrated in Fig. S2. Obviously, the TPE-peptide probes P4, P8, P9, P11 gave higher emission among the 11 probes investigated and P11 gave the highest emission intensity. MALDI-TOF-MS were performed to investigate the adducts formed after reaction of OPs with peptide probes. As shown in Fig. 1, the m/z signals of P4, P8, P9, P11 appeared at 1680.36, 1649.28, 1663.36, 1621.87, respectively. After reacted with OPS, the m/z signals of P4, P8, P9, P11 appeared at 1788.23, 1757.56, 1771.52, 1729.04, respectively. Indicating the molecular weight change of 108 was due the adduction of PO3C2H6-[H] to the peptides. Other probes (P1, P5, P6, P7, P10) have weak ability to bind OPs and weak m/z signals were observed for the adducts formed between OPs and peptide P1, P5, P6, P7, P10, respectively (Fig. S3). From these results, it can be inferred that TPE-Peptide with the higher ability to from adducts with OPs would have higher fluorescence intensity. 3.3. AIE condition optimization The reaction conditions including pH (pH 3, 5, 7, 9, 10), temperature (4, 25, 37, 55, 70 ℃) and time (0, 5, 15, 30, 45, 60 min) of TPEPeptide probe reaction with OPs (TPE-Peptide-OPs) were also investigated (Fig. S1). The probe showed weak fluorescence intensity at lower pH (pH ≤ 5) and this might be due to the electron-withdrawing properties of protonated imidazolyl, amino and phenol groups (Yang et al., 2013; Moyer et al., 2014). The highest signal enhancement was found at pH 7.0. The effects of temperature on TPE-Peptide-OPs’ emission was shown in Fig. S1C. The optimal temperature of probe P4, P8 and P11 was at 37
Fig. 1. MALDI-mass spectrum of active TPE- peptide probes (A, B, C, D) without OPs and with OPs. 4
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Fig. 2. Negatively stained TEM images of the active probes without OPs and with OPs. Scale bar: 200 nm.
Fig. 3. AFM images of the active probes without OPs and with OPs.
aggregation, indicating that the TPE-Peptide probe bound with OPs switched on the enhanced fluorescence.
To further confirm whether the TPE-Peptide probes can turn on enhanced emission after bound with OPs or not, the probe or TPEPeptide-OPs adducts were imaged by CFM (Fig. 5). The merged images in Fig. 5 show that the enhanced emission was obtained only after
Fig. 4. Circular dichroism spectroscopy (CD) measurements of the active probes without (A) and with OPs (B). 5
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Fig. 5. Ultra high resolution fluorescence confocal images of the active probeswithout and with OPs from the same area under bright field (B) and fluorescence (F). The merged image showed that fluorescence was observed from aggregate only, suggesting that the TPE-Peptide binds on OPs to “switch-on” fluorescence.
pH 7.0 for 15 min (Fig. 6). Standard addition method was used in order to study the accuracy of the TPE-Peptide probe technique in detection of OPs in real samples. 25, 50, 75, 100 μM were selected as labeling levels and tested for quantitative analysis of the methods and their recovery was found to be ranged from 85.34%–96.25 % (Table S1). Real samples carried with OPs were detected by TPE-Peptide probe technique and there were 87.65 μM methyl paraoxon, 52.89 μM methyl parathion and 65.01 μM ethyl paraoxon, respectively. These samples were also analyzed by GC–MS to investigate the precision of the developed TPE-Peptide probe method (Fig. 6), and the concentrations read by GC–MS were 86.40, 52.04 and 63.84 μM, respectively (Fig. S7). The little variance (< 2 %) indicated the high precision of this TPEPeptide probe method (Table S2). The detection limit were 0.61 μM (15.07 μg/kg, methyl paraoxon), 0.57 μM (14.07 μg/kg, methyl parathion), 0.60 μM (14.84 μg/kg, ethyl paraoxon), respectively, which were lower than the allowable maximum pesticide residue limits of European Union (0.05 mg/kg). It should be noted that at present, the
3.5. Binding stoichiometry The I/I0 (the emission intensity I, in presence of TPE-Peptide-OPs adducts; I0, in absence of OPs) value of TPE-Peptide probe P11 was studied with the probe concentrations ranged from 0 to 70 μM and I/I0 reached highest value at 50 μM. So the TPE-Peptide probe concentration of 50 μM was used in the detection of OPs. As shown in Fig. S6, the highest emission was observed at a 0.5 mol fraction of OPs, indicating that TPE-Peptide may form a 1:1 adduct with OPs and this result was correlated with the MALDI-TOF-MS results (Fig. 1). 3.6. Real sample analysis Samples of cabbage spiked with different concentrations of OPs were processed according to the NY/T 761–2008 and then detected with fluorescent TPE-Peptide probe method after incubation in 37 ℃ at
Fig. 6. OPs can be quantified according to the fluorescence spectrum and a linear working range was constructed for OPs. Fluorescence standard curve of the actual samples of methyl paraoxon (A), ethyl paraoxon (B), methyl parathion (C) with varying concentration. Fluorescence spectrum of the actual samples of methyl paraoxon (a), ethyl paraoxon (b), methyl parathion (c) with varying concentration. 6
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Declaration of Competing Interest There are no conflicts to declare. Acknowledgments This work was supported by Natural Science Foundation of China (Funding, 21575102) and Tianjin Scientific Program (18ZYPTJC00020) and the Open Funds of the State Key Laboratory of Electroanalytical Chemistry (SKLEAC201911). Dr. Haibo Li, Dr. Min Hong and Prof. Lei Wang (Liaocheng University) are thanked for collaboration. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.jhazmat.2020.122074. Fig. 7. Fluorescence intensity of the active probes (P4, 8, 9 and 11) in the presence of OPs (methyl paraoxon, ethyl paraoxon, methyl parathion) and carbamate pesticides (carbaril, methomyl).
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TPE-Peptide probe method can be used to detect the total amount of OPs, but cannot distinguish the specific types of pesticides. 3.7. Sensing of carbamate pesticides by TPE-Peptide probe method Carbamate pesticides also inhibit AChE’s activity and sensing of carbamate pesticides were also investigated using this TPE-Peptide probe method. The active probes P4, P8, P9 and P11were studied for their emission properties before and after they reacted with carbamate pesticides and the reults were shown in Fig. 7. Compared with the OPs’ sensing, TPE-Peptide probe method was less sensitive to carbaryl and methomyl investigated here. This difference may originate from the less hydrophobicity of the TPE-Peptide-carbamate than TPE-Peptide-OPs adducts because that carbaryl and methomyl have less hydrophobic groups (methyl, ester groups) than OPs (Table S3). 4. Conclusions To summarize, we developed a fluorescent TPE-Peptide probes for OPs’ detection by conjugation of AIE molecule with the R amino acid site of alternating hydrophilic and hydrophobic peptide LHLHLRL component and catalytic triads containing serine. The probe formed adducts with OPs and the induced aggregation caused enhancement of emission of TPE. The properties of these probes to detect OPs were investigated by a series of characterizations, including fluorescence spectroscopy, MALDI-TOF-MS, TEM, SEM, CFM, DLS, and CD. OPs can be quantified within 0.6–100 μM and the accuracy and precision of this fluorescent TPE-Peptide technique was investigated and they could meet the demand for real samples’ analysis. The fluorescent probe has the characteristics of high stability, environmental friendly and easy preparation, therefore this technique may find applications in routine assays of OPs. Authors statement Author contributions Jianying Wang has made substantial contributions to the acquisition, analysis, interpretation of the data for the work and drafting the work. Jiaying Zhang and Jing Wang have made contributions to the acquisition, analysis, interpretation of the data for the work. Guozhen Fang have made contributions to correction of the drafting the work. Jifeng Liu has made substantial contributions to the conception and design the work, revision of the draft and approved the final version to be submitted. Shuo Wang has made substantial contributions to the project administration and correction of the drafting the work. 7
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