车思莹
浙江工业大学化学工程学院
概述：设计了一种由离子液体组成的荧光探针，通过“富集和检测”策略对百草枯进行敏感检测。 灵敏度的增加源于静电吸引，阴离子像一个巨大的夹持器一样夹持阳离子百草枯。
A Fluorescent and Colorimetric Ionic Probe Based on Fluorescein for the Rapid and Onsite Detection of Paraquat in Vegetables and Environment
Siying Che, [a] Xiutan Peng, [a] Yiwan Zhuge, [a] Xinlan Chen, [a] ChunSong Zhou, [a] Haiyan Fu,*[b] Yuanbin She.*[a]
[a] College of Chemical Engineering, Zhejiang University of Technology, Hangzhou, 310014, China
[b] College of Pharmacy, South-Central University for Nationalities, Wuhan 430074, China
ABSTRACT: Detection of pesticide paraquat is of considerable significance to ensure food safety and its rapid and onsite detection is still a challenge. Aimed at ion characteristics of paraquat, an "enrichment and detection" strategy was proposed to improve sensitivity through electrostatic attractions and ion character of probes was adopted to increase portability through avoiding aggregation-caused quenching effects in the paper strips. Herein, a novel anion-functionalized ionic liquids probe with a large conjugated plane and rich π-electrons ([Fluo][P66614]2) was designed as a fluorescent and colorimetric dual-channel probe to sensitively and rapidly detect trace amounts of paraquat in vegetables and the environment. The proposed probe exhibited good linearity with a detection limit of 64.0 nM in the paraquat concentration range of 0.3-7.0 μM (fluorometry) and 0.1 μM in that of 0.1-8.0 μM (colorimetry), respectively. And it displayed a rapid fluorescence quenching response from green to dark (< 5 s) and excellent anti-interference (among 23 other pesticides) due to dual effects of electrostatic attraction and π-π stacking. Most importantly, the lipophilic ionic liquid probe could be applied in real vegetables and environment samples with a satisfying recovery rate of 98-103% and assembled into a handy paper strip that achieved the visual semi-quantitative detection of paraquat. This ionic probe provides a feasible approach for rapidly and conveniently detecting paraquat for ensuring agricultural and food safety and opens a new avenue to detect ion-responsive analytes in real complex samples by an "enrichment and detection" strategy.
KEYWORDS: ionic liquids, fluorescent probes, colorimetric probes, paraquat detection, paper strips
1. INTRODUCTION
Paraquat (PQ) is a highly effective and non-selective herbicide that is widely used for weed control in agriculture and gardening practices around the world. The improper usage of paraquat inevitably leads to excess residues in agricultural products and ecological environment.1-3 Paraquat is extremely toxic to humans and can be absorbed via skin exposure, breathing, and the digestive tract, which can lead to nerve damage, multiple organ failure, and even death.4-6 Due to these risks, paraquat residues in agricultural products and the ecological environment must be strictly specified and monitored. The China Food Safety Organization has set residual restrictions on paraquat. For example, the maximum dose of paraquat is approximately 0.05 mg/kg in leafy vegetables, and 0.01 mg/kg in pulp fruit (GB 2763-2021). Besides, the U.S. Environmental Protection Agency (USEPA) limit of paraquat residues is 200 μg / L (7.8 × 10-7 M) in drinking water.7,8 At present, there are many methods to detect pesticides, such as gas chromatography-mass spectrometry (GC-MS),9,10 liquid chromatography-mass spectrometry (LC-MS),10,11 surface-enhanced Roman spectroscopy (SERS),12,13 and electrochemical methods.14,15 Although these methods are sensitive and accurate, they require expensive instruments, complex pretreatment, or highly trained technicians, making them unsuitable for low-cost and on-site analysis. Thus, it is essential to develop a rapid, simple, and portable analysis method for real-time and on-site monitoring paraquat.
Due to easy operation, high sensitivity, and visual detection, optical chemical sensors have become the preferred materials for the analysis of pesticides. However, most of the typical optical sensors rely on incubations of enzymes, which greatly increased detection time.16-19 For instance, Lin's research group20 relied on enzyme inhibition caused by organophosphorus pesticides (OPs) for detection of OPs and achieved good results, where the whole process took over an hour. Besides, owing to the limitations of the properties of enzymes, it is difficult to load to solid-state sensors such as paper platforms to achieve portable detection. Simultaneously, conventional fluorescent materials are prone to induce aggregation-caused quenching (ACQ) effects and are not suitable for loading.21,22 To avoid the ACQ effects, Flood et al. came up with a general method to transfer optical properties of a fluorophore to the crystalline state by formulating cationic dyes into ionic lattices, where ion-sequestering lattices (SMILES) could perfectly retain the dye’s optical properties in solids.23 Interestingly, novel materials ionic liquids (ILs) as chemosensors can avoid restriction of enzymes use, shorten measurement time and act similarly as a class of ionized dyes unaffected by ACQ. ILs are environmentally friendly materials that are usually in the liquid state at room temperature24,25 and becoming more attractive in analytical field due to high tunability and unique optical properties.26-28 In 2019, Tao’s group29 designed a biocompatible curcumin IL to directly analyze real explosives within only 5 s, suggesting that the IL exhibited a fast response to analytes. Subsequently, Wang et al.30 demonstrated a universal approach that the fluorenscence of ILs could be significantly enhanced by the thermally activated delayed fluorescence process. Above those researches, it is promising to develop a kind of IL probe for rapid and portable detection of paraquat, as long as the tunability of ILs is fully utilized.
"Enrichment and detection" strategies were adopted in design of a IL probe to achieve high sensitivity, fast and on-site portable detection of paraquat. First, it was screened of an anion-functionalized IL probe with a large conjugated plane and rich π-electrons for paraquat detection. This anion-functionalized probe could have a strong electrostatic attraction with the cationic paraquat, and this "enrichment" can exclude the vast majority of uncharged pesticides and narrow the distance between the paraquat and the probe, providing a prerequisite for specific and rapid detection of paraquat.31,32 Immediately after, fluorescein was selected as a fluorophore of the probe to offer optical signal change due to high fluorescence efficiency, rich conjugated π-electrons, and vivid colors.33,34 Its fluorescence would be quenched after addition with paraquat because the electron poor π-cloud pyridine aromatic ring of paraquat is prone to electron transfer and π-π stacking.35-37 In addition, the charge effect and signal amplification of such ILs also efficiently made up for the lack of specificity and sensitivity of existing optical probes.38 Secondly, lipophilic ionic liquids can liquefy solid dye molecules with rigid chemical structures, which effectively avoid ACQ effects through spatial and electronic isolation and offer the possibility to serve as a paper-based probe with efficient fluorescence emission.39,40
In this work, a fluorescein-based anion-functionalized ionic liquid probe, [Fluo][P66614]2 (the probe’s name abbreviated as FIL), was designed for the fluorescence and colorimetry of paraquat detection (Scheme 1). The FIL exhibited sensitively green fluorescence quenching with the addition of paraquat through electrostatic attraction and π-π stacking dual effects, and the limit of detection (LOD) reached 64.0 nM in the linear range of 0.3-7.0 μM. It also displayed apparent color changes from green to pink under daylight, enabling visible to the naked eye with an LOD of 0.1 μM in the range of 0.1-8.0 μM. Moreover, the probe had good selectivity and anti-interference ability, which could distinguish paraquat from 23 other pesticides. Most importantly, portable paper strips were prepared through a one-step dip-dyeing method and successfully applied in real samples. The ion properties of IL not only improved the sensitivity for paraquat detection through electrostatic attraction but also enhanced the portability by avoiding ACQ effects in the paper strips, providing a convenient, effective and feasible strategy for paraquat in on-site and in-time detection.
Scheme 1. Schematic Illustration for Detection of PQ by Fluorescent and Colorimetric Signals
2. MATERAILS AND METHODS
2.1. Reagents and Instruments. Fluorescein (90%), acetic acid (99.5%), triazophos (> 98%), pentachloronitrobenzene (95%), londax (analytical standard), deltamethrin (≥ 98%), trifluralin (98%), phoxim (99%), acephate (99%), acetochlor (analytical standard), profenofos (analytical standard), fomesafen (99.5%), pretilachlor (97.5%), diazinon (98%), chlorpyrifos (99%), and Amberlite 717 were purchased from Aladdin Biochemical Technology Co., Ltd. Trihexyl (tetradecyl) phosphonium chloride (97%), aclonifen (analytical standard), and machette (analytical standard) were purchased from J&K Scientific, Ltd. Paraquat dichloride (98%) and carbendazim (98%) were purchased from Macklin Biochemical Co., Ltd. Cyfluthrin, beta-cypermethrin and dichlorvos analytical standards were purchased from Accustandard. Diquat dibromide, Difenzoquat, mepiquat and chlormequat solutions (100 μg / mL in ethanol) were purchased from Rhawn Reagent Co., Ltd. Ethanol (≥ 99.7%) was purchased from General-Reagent. Above chemicals were purchased directly for use and dissolved in ethanol.
1H NMR spectra of chemicals in DMSO-d6 were recorded on a Bruker 400 MHz spectrometer. FT-IR spectra were obtained using a Thermo Scientific IN10 FT-IR spectrometer. Fluorescence and UV-visible absorption spectra were obtained via a Hitachi F-7000 spectrometer and Shimadzu UV-2600 spectrometer, respectively. Photographs were taken in daylight or in a dark box under 365 nm UV light.
2.2. Synthesis of [Fluo][P66614]2. The target FIL probe was synthesized by a one-step acid-base neutralization reaction. First of all, [P66614]OH was prepared from trihexyl (tetradecyl) phosphonium chloride ([P66614]Cl) by using an anion-exchange resin (Amberlite 717). Next, fluorescein and [P66614]OH were added into a single-necked flask at a molar ratio of 1: 2, and ethanol was added to dissolve the mixture. The mixture was stirred at 60 ℃ for 12 hours and the solvent was removed by rotary evaporation and then drying at 80 ℃ with nitrogen bubbling to acquire [Fluo][P66614]2 as a brown-red viscous liquid product. 1H NMR (400 MHz, DMSO-d6; ppm), [Fluo]- anion: δ 7.85 (d, 1H), 7.34 (t, 1H), 7.28 (t, 1H), 6.91 (d, 1H), 6.56 (s, 1H), 6.54 (s, 1H), 5.96 (d, 1H), 5.94 (d, 1H), 5.88 (s, 1H), 5.87 (s, 1H); [P66614]+ cation: proton peaks at 2.27–0.75 ppm (136H) attributed to methyl and methylene groups.
2.3. Fluorescence Detection of Paraquat. In the test system, FIL and various concentrations of paraquat or sample solution were added into centrifugation tubes, and finally adjusted the total volume to 1 mL with ethanol. The mixtures were well mixed evenly and then poured into a cuvette to measure their fluorescence spectra. The excitation light wavelength was set at 454 nm (λex = 454 nm), and the excitation and emission slit widths were both set at 5 nm. The fluorescence intensity and peak position were recorded.
2.4. Euclidean Distance (ED). According to images to obtain the color space values (RGB), the color change was determined by differences in the images between before and after a reaction:
ΔR = |R - R0|
ΔG = |G - G0|
ΔB = |B - B0|
R0/G0/B0 represents the image before the reaction while R/G/B represents the image after the reaction (red, green, and blue, respectively). ΔR/ΔG/ΔB are the differences in colors of the images. The visual response of FIL to paraquat was tracked by an overall color change which can be calculatd as the total Euclidean distance (ED):41,42
ED = [(∑ΔR2) + (∑ΔG2) + (∑ΔB2) ]1/2
2.5. Procedure for Test Strip Fabrication and Detection. The FIL-filled test strips were obtained by directly dipping the same filter paper discs (Ф = 9 cm) into FIL ethanol solution (2 mL, 2.5 × 10-5 M) everytime and then allowing them to dry in the air. And the dried test strips could be made into various shapes. When verifying the detection effect of test strips, pesticide solutions (200 μL) of different concentrations were dropped on the surface of test strips and air-dried. In addition, in practical application, a test strip was immersed into a paraquat-containing tomato extract solution, and then took out to dry. All phenomenon changes were observed under 365 nm UV light.
2.6. Preparation of Real Samples. Two water samples (river water and tap water), soils, tea leaves, lettuce, tomatoes, wheat, rice, soybeans and potatoes were collected as real samples, and a spiked recovery experiment was used to determine the recovery rate. Samples were subjected to the simple pretreatment to obtain the extracts (the experimental section in Supporting Information). Paraquat was directly added to the water samples as well as added to tea and soil extracts soaked in ethanol. Lettuce, tomatoes and potatoes were cut and added to water as well as wheat, rice and soybeans were grinded and added to ethanol to obtain their extracts, and then paraquat was added to each extract. The extracts (100 μL) was taken and added into different concentrations of paraquat and then FIL probe (100 μL) solution was added and fixed the volume with ethanol to 1 mL with final concentrations of paraquat to 5.0 × 10-7 M, 3.0 × 10-6 M and 7.0 × 10-6 M. Fluorescence spectra of the mixing solutions of all samples containing 3.2 × 10-8 M FIL and different concentrations of paraquat were determined and each set of experiments was performed in triplicate.
3. RESULTS AND DISCUSSION
3.1. Synthesis and Characterization of FIL. The FIL was synthesized by an acid-base neutralization reaction and its solution emitted a strong green fluorescence at UV light (365nm) in Figure 1a&1b. Besides, FIL exhibited a viscous liquid state at room temperature illustrating lipophilic ILs could liquefy solid fluorescein with rigid structures. The structure of FIL was characterized by 1H NMR and FT-IR spectroscopy, respectively. Compared with the 1H NMR spectrum of fluorescein, on the one hand, it could easily find that the oxhydryl proton peaks at 10.14 ppm had disappeared, which indicated fluorescein lost two protons to form the [Fluo]- anion and the residual fluorescein content was negligible (Figure S1). On the other hand, the overall spectrum of FIL shifted upfield in Figure 1c, also illustrating that the formation of the [Fluo]- anion led to an increase in the electron cloud density. Besides, the proton peaks between 2.27-0.75 ppm were assigned to methyl and methylene groups of the [P66614]+ cation, and the number of H assigned was about 136. It proved that the molar ratio of [Fluo]- and [P66614]+ was 1:2, corresponding to the ratio of ions in the probe structure. The FT-IR spectrum also supported the formation of FIL as shown in Figure S2. The frequencies of 1467 cm-1, 1596 cm-1 at fluorescein and 1463 cm-1, 1577 cm-1 at FIL belonged to the C=C stretching vibration of the aromatic ring skeleton, and the red-shifted in aromatic region (4 and 19 cm-1, respectively) might be due to the formation of an oxyanion conjugated to the benzene ring structure. In addition, the strong and broad peak at 3285 cm-1 belonged to the characteristic O-H peak in the structure of fluorescein. The disappearance of this peak in FIL indicated that fluorescein lost protons to form [Fluo]- phenoxy anion. And we observed a new peak at 1755 cm-1 attributed to stretching vibrations of C=O groups. Furthermore, the presence of [P66614]+ cations was confirmed by the appearance of new C-H stretching vibrations of methyl and methylene groups at 2926 cm-1 and 2859 cm-1 in FIL except the stretching vibrations of C-H groups of the benzene ring at 2911 cm-1. Moreover, the possible Cl- impurity content in FIL was determined by Ag+ titration. The concentration of Cl- in the [P66614][OH] should be less than 1.56 × 10-9 M since no AgCl precipitate was formed. To sum up, these data signified that the FIL probe was successfully synthesized, and in addition, the purity of FIL met analytical purity (AR) standards and was up to the requirements of probes for detecting.
A schematic illustration of paraquat detection was displayed in Scheme 1 by the FIL probe via the fluorescent and colorimetric dual-channels. [Fluo][P66614]2 indicated an obvious emission peak at 519 nm under 454 nm excitation wavelength and was observed strong green fluorescence under a UV lamp. Once the probe FIL encountered paraquat, a significant fluorescence quenching phenomenon was found near the 519 nm emission peak. In the colorimetric channel, the color of the FIL solution changed from green, orange to pink by naked eyes under daylight after adding different concentrations of paraquat. These results indicated that FIL could be used as a fluorescent and colorimetric probe for paraquat residual detection. The fluorescence intensity (F0) of FIL with an electrostatic regulation function was much greater than that of commercial fluorescein under the optimal detection conditions (Figure 1d, S3-S4), which might be attributed to the FIL signal amplification effect. ΔF/F0 was used to display the superiority of FIL in detecting paraquat. To describe quantitatively, the FIL probe exhibited the value of ΔF/F0 for paraquat of 72.6% which was far beyond that of fluorescein (11.4%). The comparison of the fluorescence quenching ratio showed that FIL obtained a higher response efficiency than the unmodified fluorescein (without charge effect), which may benefit from FIL’s special electrostatic attractions. Therefore, FIL with an electrostatic regulation function could effectively improve the fluorescence performance and sensitivity to detect paraquat.
Figure 1. (a) The chemical structure of [Fluo][P66614]2 (i.e. FIL); (b) photographs of pure FIL (viscous liquid state) and FIL ethanol solutions taken in daylight and under a 365nm UV lamp at room temperature; (c) 1H NMR spectra of fluorescein and anions of FIL (400 MHz, DMSO-d6); (d) fluorescence intensity and quenching ratio comparison of fluorescein and FIL responded to paraquat.
3.2. Properties of FIL Probe. To evaluate properties of the probe, pH, the response time and the storage time were investigated. The fluorescence spectra of FIL were measured under different pH values in Figure S5. Paraquat is stable in an acidic or neutral media43 but the fluorescence of FIL was quenched under acidic conditions, resulting in the absence of detection signals. Considering the stability of paraquat and to obtain the best detection performance, the test pH was set at 7.0. Subsequently, the response time of the probe to paraquat was investigated, as shown in Figure 2a&S6. The fluorescence of FIL was quenched within 5 s after addition of paraquat and remained unchanged afterwards, indicating the probe possessed real-time detection ability. For convenience, 15 s was chosen as the suitable response time in the following experiments. Furthermore, the probe was stored under normal temperature and pressure without any special treatment. During storage time, FIL were tested regularly and fluorescence intensity did not fluctuate much within 56 days (Figure 2b&S7), indicating that the synthesized probe had high stability and that the preservation conditions were not harsh.
2.1. Reagents and Instruments. Fluorescein (90%), acetic acid (99.5%), triazophos (> 98%), pentachloronitrobenzene (95%), londax (analytical standard), deltamethrin (≥ 98%), trifluralin (98%), phoxim (99%), acephate (99%), acetochlor (analytical standard), profenofos (analytical standard), fomesafen (99.5%), pretilachlor (97.5%), diazinon (98%), chlorpyrifos (99%), and Amberlite 717 were purchased from Aladdin Biochemical Technology Co., Ltd. Trihexyl (tetradecyl) phosphonium chloride (97%), aclonifen (analytical standard), and machette (analytical standard) were purchased from J&K Scientific, Ltd. Paraquat dichloride (98%) and carbendazim (98%) were purchased from Macklin Biochemical Co., Ltd. Cyfluthrin, beta-cypermethrin and dichlorvos analytical standards were purchased from Accustandard. Diquat dibromide, Difenzoquat, mepiquat and chlormequat solutions (100 μg / mL in ethanol) were purchased from Rhawn Reagent Co., Ltd. Ethanol (≥ 99.7%) was purchased from General-Reagent. Above chemicals were purchased directly for use and dissolved in ethanol.
1H NMR spectra of chemicals in DMSO-d6 were recorded on a Bruker 400 MHz spectrometer. FT-IR spectra were obtained using a Thermo Scientific IN10 FT-IR spectrometer. Fluorescence and UV-visible absorption spectra were obtained via a Hitachi F-7000 spectrometer and Shimadzu UV-2600 spectrometer, respectively. Photographs were taken in daylight or in a dark box under 365 nm UV light.
2.2. Synthesis of [Fluo][P66614]2. The target FIL probe was synthesized by a one-step acid-base neutralization reaction. First of all, [P66614]OH was prepared from trihexyl (tetradecyl) phosphonium chloride ([P66614]Cl) by using an anion-exchange resin (Amberlite 717). Next, fluorescein and [P66614]OH were added into a single-necked flask at a molar ratio of 1: 2, and ethanol was added to dissolve the mixture. The mixture was stirred at 60 ℃ for 12 hours and the solvent was removed by rotary evaporation and then drying at 80 ℃ with nitrogen bubbling to acquire [Fluo][P66614]2 as a brown-red viscous liquid product. 1H NMR (400 MHz, DMSO-d6; ppm), [Fluo]- anion: δ 7.85 (d, 1H), 7.34 (t, 1H), 7.28 (t, 1H), 6.91 (d, 1H), 6.56 (s, 1H), 6.54 (s, 1H), 5.96 (d, 1H), 5.94 (d, 1H), 5.88 (s, 1H), 5.87 (s, 1H); [P66614]+ cation: proton peaks at 2.27–0.75 ppm (136H) attributed to methyl and methylene groups.
2.3. Fluorescence Detection of Paraquat. In the test system, FIL and various concentrations of paraquat or sample solution were added into centrifugation tubes, and finally adjusted the total volume to 1 mL with ethanol. The mixtures were well mixed evenly and then poured into a cuvette to measure their fluorescence spectra. The excitation light wavelength was set at 454 nm (λex = 454 nm), and the excitation and emission slit widths were both set at 5 nm. The fluorescence intensity and peak position were recorded.
2.4. Euclidean Distance (ED). According to images to obtain the color space values (RGB), the color change was determined by differences in the images between before and after a reaction:
ΔR = |R - R0|
ΔG = |G - G0|
ΔB = |B - B0|
R0/G0/B0 represents the image before the reaction while R/G/B represents the image after the reaction (red, green, and blue, respectively). ΔR/ΔG/ΔB are the differences in colors of the images. The visual response of FIL to paraquat was tracked by an overall color change which can be calculatd as the total Euclidean distance (ED):41,42
ED = [(∑ΔR2) + (∑ΔG2) + (∑ΔB2) ]1/2
2.5. Procedure for Test Strip Fabrication and Detection. The FIL-filled test strips were obtained by directly dipping the same filter paper discs (Ф = 9 cm) into FIL ethanol solution (2 mL, 2.5 × 10-5 M) everytime and then allowing them to dry in the air. And the dried test strips could be made into various shapes. When verifying the detection effect of test strips, pesticide solutions (200 μL) of different concentrations were dropped on the surface of test strips and air-dried. In addition, in practical application, a test strip was immersed into a paraquat-containing tomato extract solution, and then took out to dry. All phenomenon changes were observed under 365 nm UV light.
2.6. Preparation of Real Samples. Two water samples (river water and tap water), soils, tea leaves, lettuce, tomatoes, wheat, rice, soybeans and potatoes were collected as real samples, and a spiked recovery experiment was used to determine the recovery rate. Samples were subjected to the simple pretreatment to obtain the extracts (the experimental section in Supporting Information). Paraquat was directly added to the water samples as well as added to tea and soil extracts soaked in ethanol. Lettuce, tomatoes and potatoes were cut and added to water as well as wheat, rice and soybeans were grinded and added to ethanol to obtain their extracts, and then paraquat was added to each extract. The extracts (100 μL) was taken and added into different concentrations of paraquat and then FIL probe (100 μL) solution was added and fixed the volume with ethanol to 1 mL with final concentrations of paraquat to 5.0 × 10-7 M, 3.0 × 10-6 M and 7.0 × 10-6 M. Fluorescence spectra of the mixing solutions of all samples containing 3.2 × 10-8 M FIL and different concentrations of paraquat were determined and each set of experiments was performed in triplicate.
3. RESULTS AND DISCUSSION
3.1. Synthesis and Characterization of FIL. The FIL was synthesized by an acid-base neutralization reaction and its solution emitted a strong green fluorescence at UV light (365nm) in Figure 1a&1b. Besides, FIL exhibited a viscous liquid state at room temperature illustrating lipophilic ILs could liquefy solid fluorescein with rigid structures. The structure of FIL was characterized by 1H NMR and FT-IR spectroscopy, respectively. Compared with the 1H NMR spectrum of fluorescein, on the one hand, it could easily find that the oxhydryl proton peaks at 10.14 ppm had disappeared, which indicated fluorescein lost two protons to form the [Fluo]- anion and the residual fluorescein content was negligible (Figure S1). On the other hand, the overall spectrum of FIL shifted upfield in Figure 1c, also illustrating that the formation of the [Fluo]- anion led to an increase in the electron cloud density. Besides, the proton peaks between 2.27-0.75 ppm were assigned to methyl and methylene groups of the [P66614]+ cation, and the number of H assigned was about 136. It proved that the molar ratio of [Fluo]- and [P66614]+ was 1:2, corresponding to the ratio of ions in the probe structure. The FT-IR spectrum also supported the formation of FIL as shown in Figure S2. The frequencies of 1467 cm-1, 1596 cm-1 at fluorescein and 1463 cm-1, 1577 cm-1 at FIL belonged to the C=C stretching vibration of the aromatic ring skeleton, and the red-shifted in aromatic region (4 and 19 cm-1, respectively) might be due to the formation of an oxyanion conjugated to the benzene ring structure. In addition, the strong and broad peak at 3285 cm-1 belonged to the characteristic O-H peak in the structure of fluorescein. The disappearance of this peak in FIL indicated that fluorescein lost protons to form [Fluo]- phenoxy anion. And we observed a new peak at 1755 cm-1 attributed to stretching vibrations of C=O groups. Furthermore, the presence of [P66614]+ cations was confirmed by the appearance of new C-H stretching vibrations of methyl and methylene groups at 2926 cm-1 and 2859 cm-1 in FIL except the stretching vibrations of C-H groups of the benzene ring at 2911 cm-1. Moreover, the possible Cl- impurity content in FIL was determined by Ag+ titration. The concentration of Cl- in the [P66614][OH] should be less than 1.56 × 10-9 M since no AgCl precipitate was formed. To sum up, these data signified that the FIL probe was successfully synthesized, and in addition, the purity of FIL met analytical purity (AR) standards and was up to the requirements of probes for detecting.
A schematic illustration of paraquat detection was displayed in Scheme 1 by the FIL probe via the fluorescent and colorimetric dual-channels. [Fluo][P66614]2 indicated an obvious emission peak at 519 nm under 454 nm excitation wavelength and was observed strong green fluorescence under a UV lamp. Once the probe FIL encountered paraquat, a significant fluorescence quenching phenomenon was found near the 519 nm emission peak. In the colorimetric channel, the color of the FIL solution changed from green, orange to pink by naked eyes under daylight after adding different concentrations of paraquat. These results indicated that FIL could be used as a fluorescent and colorimetric probe for paraquat residual detection. The fluorescence intensity (F0) of FIL with an electrostatic regulation function was much greater than that of commercial fluorescein under the optimal detection conditions (Figure 1d, S3-S4), which might be attributed to the FIL signal amplification effect. ΔF/F0 was used to display the superiority of FIL in detecting paraquat. To describe quantitatively, the FIL probe exhibited the value of ΔF/F0 for paraquat of 72.6% which was far beyond that of fluorescein (11.4%). The comparison of the fluorescence quenching ratio showed that FIL obtained a higher response efficiency than the unmodified fluorescein (without charge effect), which may benefit from FIL’s special electrostatic attractions. Therefore, FIL with an electrostatic regulation function could effectively improve the fluorescence performance and sensitivity to detect paraquat.
Figure 1. (a) The chemical structure of [Fluo][P66614]2 (i.e. FIL); (b) photographs of pure FIL (viscous liquid state) and FIL ethanol solutions taken in daylight and under a 365nm UV lamp at room temperature; (c) 1H NMR spectra of fluorescein and anions of FIL (400 MHz, DMSO-d6); (d) fluorescence intensity and quenching ratio comparison of fluorescein and FIL responded to paraquat.
3.2. Properties of FIL Probe. To evaluate properties of the probe, pH, the response time and the storage time were investigated. The fluorescence spectra of FIL were measured under different pH values in Figure S5. Paraquat is stable in an acidic or neutral media43 but the fluorescence of FIL was quenched under acidic conditions, resulting in the absence of detection signals. Considering the stability of paraquat and to obtain the best detection performance, the test pH was set at 7.0. Subsequently, the response time of the probe to paraquat was investigated, as shown in Figure 2a&S6. The fluorescence of FIL was quenched within 5 s after addition of paraquat and remained unchanged afterwards, indicating the probe possessed real-time detection ability. For convenience, 15 s was chosen as the suitable response time in the following experiments. Furthermore, the probe was stored under normal temperature and pressure without any special treatment. During storage time, FIL were tested regularly and fluorescence intensity did not fluctuate much within 56 days (Figure 2b&S7), indicating that the synthesized probe had high stability and that the preservation conditions were not harsh.
Figure 2. (a) Time response tests toward PQ and (b) fluorenscence stability of the probe during storage time.
3.3. Fluorescence and Colorimetric Response of FIL Probe to Paraquat. In order to evaluate the sensitivity of the FIL probe for paraquat, gradient experiments with various concentrations of paraquat were conducted under the optimal conditions using a fluorescence method. In Figure 3a, as the concentration of paraquat increased, the degree of fluorescence quenching also increased. The relationship between the change in fluorescence intensity (ΔF = F0 - F) and the paraquat concentration is shown in the inset. Changes in the linear range (3.0 × 10-7 to 7.0 × 10-6 M) were divided into two sections, and the point at 2.5 × 10-6 M was the demarcation point where the linear correlation equations were ΔF = 68.61C + 621.4 (R2 = 0.9958) and ΔF = 323.1C (R2 = 0.9974), respectively. The limit of detection (LOD) for paraquat was 6.4 × 10-8 M (64.0 nM) based on three replicates and the standard deviation (LOD = 3σ/K, where σ is the standard deviation of the blank system, and K is the slope of the calibration curve), which was lower than the USEPA residue limit value of 7.8 × 10-7 M. Recent reported researches about paraquat optical detection were summarized in Table S1. Considering response time, the linear range, LOD, selectivity, RSD and acuuracy six factors, this FIL probe exhibited satisfactory performance. Figure 3b exhibited the photographs of FIL at various concentrations of paraquat and a clear visual change from bright green to colorless was observed by naked eyes under a 365 nm UV lamp.
Simultaneously, the response of FIL probe in the presence of paraquat was tested by colorimetry under daylight and UV-Vis absorption spectra. A series of FIL solutions containing various concentrations of paraquat displayed a distinct color change from green, orange and pink with the naked eye (Figure 3c). The UV-Vis spectra, in Figure S8a, manifested that the multiple visible colors were attributed to the change in absorbed colors (wavelength range at 450-500 nm), especially the increased absorption of light at 500 nm brought about the solution color moving from green more towards pink. To describe quantitatively, the total Euclidean distance (ED) was adopted to track the overall color change of response of FIL to paraquat. It showed a good linearity in the paraquat concentration range of 1.0 × 10-7-8.0 × 10-6 M (Figure S8b) and the correlation equation was calculated to be ED = 6.518C + 12.37 (R2 = 0.9981). The LOD for the visual detection of paraquat was 0.1 μM. To more clearly elucidate the apparent changes in color, the CIELab color model was introduced.44 In accordance with the model, the parameters a* and b* for FIL with paraquat (Figure 3c) and fluorescein with paraquat (Figure S9) were measured at the same lightness (L). Then, the corresponding points were connected as presented in Figure 3d. With concentrations of paraquat increased from 0 to 8.0 μM, the a* value of FIL-PQ increased from -16 to +6 while the b* value decreased from 41 to 8; Similarly, the a* value of Fluorescein-PQ increased from -1 to +4 while the b* value decreased from 7 to 0. By comparison, the red-line segment was longer and distributed in different color areas, which implied that the FIL manifested a wider range of discoloration. All of these evidences declared that such a novel colorimetric probe with a high sensitivity and rapid response owned great potential applications for monitoring paraquat residues by naked eyes.
Figure 3. (a) Fluorescence emission spectra of the solutions of 3.2×10-8 M FIL probe with various concentrations of PQ (0, 0.3, 0.5, 0.7, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 μM (λex = 454 nm)); the inset showed the linear relationship between fluorescence intensity change (ΔF) and PQ concentration; (b) the photographs of the FIL (6.3 × 10-6 M) with various concentrations of PQ (from left to right: 0, 0.1, 1.0, 4.0, 5.0, 6.0, 8.0, 10.0, 50.0, and 100.0 μM) under 365 nm UV light and (c) in daylight (from left to right: 0, 0.1, 1.0, 4.0, 5.0, 6.0, and 8.0 μM); (d) color parameters of FIL and fluorescein with addition of various concentrations of PQ (red line: a* and b* of FIL-PQ; blue line: a* and b* of fluorescein-PQ) on the CIELab color model.
3.4. Selectivity Study. The selectivity is an essential metric of probe. To evaluate the selectivity of FIL, we tested interference of 19 potential pesticides through parallel experiments, and the fluorescence emission spectra were recorded in Figure S10. Under the same conditions, only paraquat produced significant fluorescence quenching behavior on FIL while other interfering pesticides had almost no changes in their fluorescence intensity in Figure 4. Particularly, based on ionic properties, the same parallel experiments were again performed on five cationic types of pesticides, including three herbicide pesticides (paraquat, diquat and difenzoquat) and two plant growth regulators (chlormequat & mepiquat) in Figure S11. Only the addition of paraquat changed the FIL solution’s color from green to orange and the severe quenching of green fluorescence, when other cationic pesticides did not occur. These results manifested that FIL could specifically recognize paraquat. Likewise, competition experiments were carried out to further illustrate its anti-interference performance under the same conditions, where paraquat and equal equivalent other pesticides were mixed together for fluorescence detection. None of the interfering pesticides led to distinct fluorescence quenching for FIL and mixed pesticides caused significant quenching phenomena only in the presence of paraquat. Based on the above results, FIL processed specific recognition to paraquat, and was inert to the other 23 kinds of pesticides.
Figure 4. The fluorescence intensity of 3.2×10-8 M FIL with the addition of 5.0 × 10-6 M interfering pesticides in the presence and absence of 5.0 × 10-6 M PQ after 15 s.
3.5. Sensing Mechanism. In order to confirm a possible response mechanism, the interaction between paraquat and FIL was investigated via DFT calculations, UV-Vis, the fluorescence lifetime, and 1H NMR experiments. Figure 5a showed the optimized structure calculated by DFT calculations at B3LYP/6-31+(d) level and the distance between paraquat and FIL molecules was predicted to be 2.700-3.426 Å. To reveal molecular interactions, the Mulliken charge distribution and electrostatic potential map (ESP) were predicted as shown in Figure S12a&5a. The Mulliken charge distribution displayed that O atoms of FIL tended to donate electrons, while N atoms of paraquat involved in binding to FIL tended to gain electrons. Meantime, it could be noted in the ESP image of the FIL-PQ complex that it was a compound whose color ranged from dark red to blue. There was a strong polarization effect between the FIL anion and the paraquat cation molecules, indicating an electrostatic attraction could occur between the them as a driving force for response.37 In addition, the increased dihedral angle of the paraquat molecule tended to planarize the molecule (from 139.4° to 169.6°) due to the electrostatic attraction effect, which was more conducive to the formation of weak π-π stacking with the FIL’s π-electron conjugated system (Figure S12b). It was also studied by UV-Vis spectra (Figure 5b), in which the π→π* transition caused by FIL was concentrated at 200-250 nm. As the increasing concentrations of paraquat addition, the absorption peaks at 225 nm occurred a clear red shift which implied a decrease of the π→π* transition energy. This could be speculated that the π-conjugated system was expanded owing to the formation of a new ground-state complex by the π-π stacking between FIL and paraquat.45,46 The fluorescence lifetime of the probe remained almost constant before and after the response, further suggesting the interaction was attributed to a static quenching process in Figure 5c. Besides, by comparing the 1H NMR spectra of paraquat, FIL, and the FIL-PQ complex in Figure 5d, signal peak changes of the FIL-PQ complex were observed. The H peaks corresponding to FIL significantly shifted downfield, and the H peaks of the pyridine aromatic ring on paraquat separated and shifted upfield. This might be attributed to the good π-π interactions and electron transfer between the aromatic groups, which changed the electron cloud density. The above data demonstrated that paraquat were attracted to the anion of FIL by electrostatic interaction and further quench fluorescence of FIL through π-π stacking interactions.
Figure 5. (a) The ESP image of the optimized structure of FIL-PQ complex (blue: positive potentials, red: negative potentials); (b) the absorption spectra of FIL in the absence and presence of various concentrations of PQ; (c) the fluorescence decay curves of FIL and FIL-PQ; (d) 1H NMR spectra of PQ, FIL, and FIL-PQ (400 MHz, DMSO-d6).
3.6. Practical Applications. Ten real samples were collected for recovery experiments to evaluate the practical application ability of the probe, like river water, tap water, soils, tea leaves, lettuce, tomatoes, wheat, rice, potatoes and soybeans. Thereinto, in Figure S13, it could be seen in the fluorescence emission spectra of probe solutions containing different proportions of water that a small amount of water (less than 20%) had a ignorable effect on the FIL detector system. HPLC analysis was used to record the initial concentration of paraquat in all samples and the results indicated that paraquat was not detected in all the samples, which meant that the original paraquat in the real sample was negligible (Figure S14). Then, the real samples were spiked with different concentrations of paraquat solutions, and the sensing performance of the probe was recorded in Figure S15 and Table 1. In three repeated experiments for each real sample, the relative standard deviation (RSD) of these data was 0.18%-3.10% as well as the relative error (Er) was 0.07%-3.39%, indicating these data behaved high precision and satisfactory accuracy. That was to say that the FIL-based probe achieved satisfactory recoveries (98-103%), reproducibility and good analytical accuracy (verified by HPLC analysis). Furthermore, statistical analysis were performed by IBM SPSS Statistics and the P value calculated as 0.99 between the fluorescence and the HPLC method. And the P > 0.05 was considered that the data were not found significant difference between the two methods. These results demonstrated that the probe had potential value for paraquat residues detection in real samples including food samples and environment examples.
Table 1. Recovery Results of Paraquat in Real Samples.
3.7. Portable Test strips. For convenience and portability, a test strip was prepared. As shown in Figure 6a, test strips were obtained through a facile single-step method, in which filter paper was dyed in a constant concentration FIL solution and then dried naturally. Paper strips were processable and could be folded into the desired shape according to actual demands, such as a crane (Figure 6d). The test strips were characterized by scanning electron microscopy (SEM) images and energy dispersive X-ray spectroscopy (EDS) mapping. Compared with the untreated filter paper, the SEM images showed that the micromorphology of the fibers was undamaged, and the magnified SEM image showed that the probe molecules were well-distributed on the fiber surface (Figure S16). The EDS mapping displayed the atomic percentage (At%) of phosphorus element from 0 to 0.07 after dipping, confirming that the probe molecules were transferred onto the fibers (Figure S17 and Table S2). Firstly, the test strip test was carried by the addition of six different types of pesticides (paraquat, triazophos, bensulfuron methyl, carbendazim, cyfluthrin, aclonifen) with various concentrations. The structures of these pesticides are shown in Figure S18. As expected, in Figure 6b, these other pesticides did not cause significant fluorescence color change while only paraquat darkened green fluorescent color of paper stripes, which indicated that the test strip had good selectivity for paraquat. When different concentrations of paraquat were added to the test papers (Figure 6c), the fluorescence color gradually darkened from green, and the lowest detection concentration by naked eyes was as low as 1.0 × 10-7 M, which was lower than the USEPA limit (7.8 × 10-7 M). Then the paper strips were further applied in tomatoes as real samples. six kinds of different pesticides were added tomato extract solutions in Figure S19, 6e. The test strip changed from the original green to a darker color after exposure to paraquat, while the other test strips basically did not change. These evidences revealed that the proposed test strip device holds great promise for on-site and quantitative identification of paraquat in real food samples, benefited from easy manufacture, high stability, and portability.
Figure 6. (a) The preparation of a test strip and the experimental process for detecting PQ in tomatoes; photos of the emission under UV light (365 nm); (b) the response of different pesticides at different concentrations (1.0 × 10-4 M, 1.0 × 10-5 M, and 1.0 × 10-6 M) to the test strips; (c) the response when adding different concentrations of paraquat, including 0, 0.1, 0.5, 1.0, 3.0, 5.0, 8.0, 10.0, 50.0, and 100.0 μM, respectively; (d) the photographs of the test paper folded into a paper crane under daylight and a 365 nm UV light, in which the head was stained with the FIL probe solution; (e) the test strip was used to detect 1.0 × 10-5 M pesticides on tomatoes (from left to right: PQ; ethanol; 1-5 were triazophos, bensulfuron methyl, carbendazim, cyfluthrin, and aclonifen, respectively).
Simultaneously, the response of FIL probe in the presence of paraquat was tested by colorimetry under daylight and UV-Vis absorption spectra. A series of FIL solutions containing various concentrations of paraquat displayed a distinct color change from green, orange and pink with the naked eye (Figure 3c). The UV-Vis spectra, in Figure S8a, manifested that the multiple visible colors were attributed to the change in absorbed colors (wavelength range at 450-500 nm), especially the increased absorption of light at 500 nm brought about the solution color moving from green more towards pink. To describe quantitatively, the total Euclidean distance (ED) was adopted to track the overall color change of response of FIL to paraquat. It showed a good linearity in the paraquat concentration range of 1.0 × 10-7-8.0 × 10-6 M (Figure S8b) and the correlation equation was calculated to be ED = 6.518C + 12.37 (R2 = 0.9981). The LOD for the visual detection of paraquat was 0.1 μM. To more clearly elucidate the apparent changes in color, the CIELab color model was introduced.44 In accordance with the model, the parameters a* and b* for FIL with paraquat (Figure 3c) and fluorescein with paraquat (Figure S9) were measured at the same lightness (L). Then, the corresponding points were connected as presented in Figure 3d. With concentrations of paraquat increased from 0 to 8.0 μM, the a* value of FIL-PQ increased from -16 to +6 while the b* value decreased from 41 to 8; Similarly, the a* value of Fluorescein-PQ increased from -1 to +4 while the b* value decreased from 7 to 0. By comparison, the red-line segment was longer and distributed in different color areas, which implied that the FIL manifested a wider range of discoloration. All of these evidences declared that such a novel colorimetric probe with a high sensitivity and rapid response owned great potential applications for monitoring paraquat residues by naked eyes.
Figure 3. (a) Fluorescence emission spectra of the solutions of 3.2×10-8 M FIL probe with various concentrations of PQ (0, 0.3, 0.5, 0.7, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 μM (λex = 454 nm)); the inset showed the linear relationship between fluorescence intensity change (ΔF) and PQ concentration; (b) the photographs of the FIL (6.3 × 10-6 M) with various concentrations of PQ (from left to right: 0, 0.1, 1.0, 4.0, 5.0, 6.0, 8.0, 10.0, 50.0, and 100.0 μM) under 365 nm UV light and (c) in daylight (from left to right: 0, 0.1, 1.0, 4.0, 5.0, 6.0, and 8.0 μM); (d) color parameters of FIL and fluorescein with addition of various concentrations of PQ (red line: a* and b* of FIL-PQ; blue line: a* and b* of fluorescein-PQ) on the CIELab color model.
3.4. Selectivity Study. The selectivity is an essential metric of probe. To evaluate the selectivity of FIL, we tested interference of 19 potential pesticides through parallel experiments, and the fluorescence emission spectra were recorded in Figure S10. Under the same conditions, only paraquat produced significant fluorescence quenching behavior on FIL while other interfering pesticides had almost no changes in their fluorescence intensity in Figure 4. Particularly, based on ionic properties, the same parallel experiments were again performed on five cationic types of pesticides, including three herbicide pesticides (paraquat, diquat and difenzoquat) and two plant growth regulators (chlormequat & mepiquat) in Figure S11. Only the addition of paraquat changed the FIL solution’s color from green to orange and the severe quenching of green fluorescence, when other cationic pesticides did not occur. These results manifested that FIL could specifically recognize paraquat. Likewise, competition experiments were carried out to further illustrate its anti-interference performance under the same conditions, where paraquat and equal equivalent other pesticides were mixed together for fluorescence detection. None of the interfering pesticides led to distinct fluorescence quenching for FIL and mixed pesticides caused significant quenching phenomena only in the presence of paraquat. Based on the above results, FIL processed specific recognition to paraquat, and was inert to the other 23 kinds of pesticides.
Figure 4. The fluorescence intensity of 3.2×10-8 M FIL with the addition of 5.0 × 10-6 M interfering pesticides in the presence and absence of 5.0 × 10-6 M PQ after 15 s.
3.5. Sensing Mechanism. In order to confirm a possible response mechanism, the interaction between paraquat and FIL was investigated via DFT calculations, UV-Vis, the fluorescence lifetime, and 1H NMR experiments. Figure 5a showed the optimized structure calculated by DFT calculations at B3LYP/6-31+(d) level and the distance between paraquat and FIL molecules was predicted to be 2.700-3.426 Å. To reveal molecular interactions, the Mulliken charge distribution and electrostatic potential map (ESP) were predicted as shown in Figure S12a&5a. The Mulliken charge distribution displayed that O atoms of FIL tended to donate electrons, while N atoms of paraquat involved in binding to FIL tended to gain electrons. Meantime, it could be noted in the ESP image of the FIL-PQ complex that it was a compound whose color ranged from dark red to blue. There was a strong polarization effect between the FIL anion and the paraquat cation molecules, indicating an electrostatic attraction could occur between the them as a driving force for response.37 In addition, the increased dihedral angle of the paraquat molecule tended to planarize the molecule (from 139.4° to 169.6°) due to the electrostatic attraction effect, which was more conducive to the formation of weak π-π stacking with the FIL’s π-electron conjugated system (Figure S12b). It was also studied by UV-Vis spectra (Figure 5b), in which the π→π* transition caused by FIL was concentrated at 200-250 nm. As the increasing concentrations of paraquat addition, the absorption peaks at 225 nm occurred a clear red shift which implied a decrease of the π→π* transition energy. This could be speculated that the π-conjugated system was expanded owing to the formation of a new ground-state complex by the π-π stacking between FIL and paraquat.45,46 The fluorescence lifetime of the probe remained almost constant before and after the response, further suggesting the interaction was attributed to a static quenching process in Figure 5c. Besides, by comparing the 1H NMR spectra of paraquat, FIL, and the FIL-PQ complex in Figure 5d, signal peak changes of the FIL-PQ complex were observed. The H peaks corresponding to FIL significantly shifted downfield, and the H peaks of the pyridine aromatic ring on paraquat separated and shifted upfield. This might be attributed to the good π-π interactions and electron transfer between the aromatic groups, which changed the electron cloud density. The above data demonstrated that paraquat were attracted to the anion of FIL by electrostatic interaction and further quench fluorescence of FIL through π-π stacking interactions.
Figure 5. (a) The ESP image of the optimized structure of FIL-PQ complex (blue: positive potentials, red: negative potentials); (b) the absorption spectra of FIL in the absence and presence of various concentrations of PQ; (c) the fluorescence decay curves of FIL and FIL-PQ; (d) 1H NMR spectra of PQ, FIL, and FIL-PQ (400 MHz, DMSO-d6).
3.6. Practical Applications. Ten real samples were collected for recovery experiments to evaluate the practical application ability of the probe, like river water, tap water, soils, tea leaves, lettuce, tomatoes, wheat, rice, potatoes and soybeans. Thereinto, in Figure S13, it could be seen in the fluorescence emission spectra of probe solutions containing different proportions of water that a small amount of water (less than 20%) had a ignorable effect on the FIL detector system. HPLC analysis was used to record the initial concentration of paraquat in all samples and the results indicated that paraquat was not detected in all the samples, which meant that the original paraquat in the real sample was negligible (Figure S14). Then, the real samples were spiked with different concentrations of paraquat solutions, and the sensing performance of the probe was recorded in Figure S15 and Table 1. In three repeated experiments for each real sample, the relative standard deviation (RSD) of these data was 0.18%-3.10% as well as the relative error (Er) was 0.07%-3.39%, indicating these data behaved high precision and satisfactory accuracy. That was to say that the FIL-based probe achieved satisfactory recoveries (98-103%), reproducibility and good analytical accuracy (verified by HPLC analysis). Furthermore, statistical analysis were performed by IBM SPSS Statistics and the P value calculated as 0.99 between the fluorescence and the HPLC method. And the P > 0.05 was considered that the data were not found significant difference between the two methods. These results demonstrated that the probe had potential value for paraquat residues detection in real samples including food samples and environment examples.
Table 1. Recovery Results of Paraquat in Real Samples.
3.7. Portable Test strips. For convenience and portability, a test strip was prepared. As shown in Figure 6a, test strips were obtained through a facile single-step method, in which filter paper was dyed in a constant concentration FIL solution and then dried naturally. Paper strips were processable and could be folded into the desired shape according to actual demands, such as a crane (Figure 6d). The test strips were characterized by scanning electron microscopy (SEM) images and energy dispersive X-ray spectroscopy (EDS) mapping. Compared with the untreated filter paper, the SEM images showed that the micromorphology of the fibers was undamaged, and the magnified SEM image showed that the probe molecules were well-distributed on the fiber surface (Figure S16). The EDS mapping displayed the atomic percentage (At%) of phosphorus element from 0 to 0.07 after dipping, confirming that the probe molecules were transferred onto the fibers (Figure S17 and Table S2). Firstly, the test strip test was carried by the addition of six different types of pesticides (paraquat, triazophos, bensulfuron methyl, carbendazim, cyfluthrin, aclonifen) with various concentrations. The structures of these pesticides are shown in Figure S18. As expected, in Figure 6b, these other pesticides did not cause significant fluorescence color change while only paraquat darkened green fluorescent color of paper stripes, which indicated that the test strip had good selectivity for paraquat. When different concentrations of paraquat were added to the test papers (Figure 6c), the fluorescence color gradually darkened from green, and the lowest detection concentration by naked eyes was as low as 1.0 × 10-7 M, which was lower than the USEPA limit (7.8 × 10-7 M). Then the paper strips were further applied in tomatoes as real samples. six kinds of different pesticides were added tomato extract solutions in Figure S19, 6e. The test strip changed from the original green to a darker color after exposure to paraquat, while the other test strips basically did not change. These evidences revealed that the proposed test strip device holds great promise for on-site and quantitative identification of paraquat in real food samples, benefited from easy manufacture, high stability, and portability.
Figure 6. (a) The preparation of a test strip and the experimental process for detecting PQ in tomatoes; photos of the emission under UV light (365 nm); (b) the response of different pesticides at different concentrations (1.0 × 10-4 M, 1.0 × 10-5 M, and 1.0 × 10-6 M) to the test strips; (c) the response when adding different concentrations of paraquat, including 0, 0.1, 0.5, 1.0, 3.0, 5.0, 8.0, 10.0, 50.0, and 100.0 μM, respectively; (d) the photographs of the test paper folded into a paper crane under daylight and a 365 nm UV light, in which the head was stained with the FIL probe solution; (e) the test strip was used to detect 1.0 × 10-5 M pesticides on tomatoes (from left to right: PQ; ethanol; 1-5 were triazophos, bensulfuron methyl, carbendazim, cyfluthrin, and aclonifen, respectively).
In general, an anion-functionalized fluorescent and colorimetric ionic probe (FIL) was designed for the rapid and onsite detection of paraquat in vegetables and the environment. Excitingly, the charged ionic probe enhanced the sensitivity of detection, in which the FIL exhibited a higher fluorescence quenching rate (ΔF/F0) for paraquat from 11.4% to 72.6% compared with commercial fluorescein. The proposed probe unveiled well-behaved linearity in the paraquat concentration range of 0.3-7.0 μM (fluorometry) and 0.1-8.0 μM (colorimetry) with detection limits of 64.0 nM and 0.1 μM, respectively, which were both lower than the residue limit specified by USEPA (7.8 × 10-7 M). Besides, it displayed a rapid fluorescence quenching response (< 5 s) and excellent anti-interference (among 23 other pesticides). These excellent response properties might benefit from the electrostatic attraction between the paraquat and FIL, followed by π-π stacking to further quench the fluorescence of FIL. Besides, FIL probe was applied to 10 different real samples with a satisfying recovery rate of 98-103%, and the results were not found significant difference from those of the HPLC method (P > 0.05). Most importantly, the lipophilic ionic liquid probe behaved outstanding loading performance and could be assembled into a handy paper strip that achieved the visual semi-quantitative detection of paraquat. This work provides a feasible approach for rapidly and conveniently detecting paraquat and opens a new avenue to determine ion-responsive analytes in real complex vegetable and environment samples by an "enrichment and detection" strategy.
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