Article pubs.acs.org/JAFC
Colorimetric and Phosphorimetric Dual-Signaling Strategy Mediated by Inner Filter Effect for Highly Sensitive Assay of Organophosphorus Pesticides Rong Zhang,†,‡,§ Na Li,†,‡,§ Junyong Sun,†,‡,§ and Feng Gao*,†,‡,§ †
Laboratory of Functionalized Molecular Solids, Ministry of Education, ‡Anhui Key Laboratory of Chemo/Biosensing, and Laboratory of Optical Probes and Bioelectrocatalysis (LOPAB), College of Chemistry and Materials Science, Anhui Normal University, Wuhu 241000, P. R. China §
S Supporting Information *
ABSTRACT: We describe here a colorimetric and phosphorimetric dual-signaling strategy for sensitive assay of organophosphorus pesticides (OPPs). The principle for assay depends on the phenomenon that the phosphorescence of MnZnS quantum dots (QDs) can be dramatically quenched by Au nanoparticles (AuNPs) through the inner filter effect (IFE) and the activity of acetylcholinesterase (AChE), an enzyme that catalytically hydrolyzes acetylthiocholine to thiocholine that can be inhibited by OPPs. By virtue of the variations of absorbance and phosphorescence of the analytical system, a dual-readout assay for OPPs has been proposed. The limits of detection for different OPPs including paraoxon, parathion, omethoate, and dimethyl dichlorovinyl phosphate (DDVP) are found to be 0.29, 0.59, 0.67, and 0.44 ng/L, respectively. The proposed assay was allowed to detect pesticides in real spiked samples and authentic contaminated apples with satisfactory results, suggesting its potential applications to detect pesticides in complicated samples. KEYWORDS: organophosphorus pesticides, inner filter effect, colorimetric and phosphorimetric dual signaling, Au nanoparticles, Mn-ZnS quantum dots, acetylcholinesterase
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metric method,10−12 colorimetric assay,12,13 fiber-optic photometer,14 chemiluminescent method,15 and electrochemical analysis16 have been exploited to assay residual pesticides in different samples. These techniques have shown attractive selectivity and sensitivity, but most of them are single-signal readout and lack diversity of detection.5−11,13−16 On the other hand, some of the optical methods are not suitable for direct and rapid detection because of the background interference of sample matrixes, and therefore complex and tedious pretreatments of samples are generally required. Obviously, significant challenges remain in developing analytical systems for fast, direct, and multisignal assay of ultratrace OPP residues in real samples including contaminated agricultural products and environmental samples.1,2,12 Multisignal sensing strategy in an analytical system has attracted more and more interest because multiple transduction models can be read out for sensing the target analyte and therefore provide improved analytical accuracy and diversity.12,17,18 To fabricate multimodel sensing through various transduction models, extracting multiple information from a single analytical system is still challenging.12,17,18 In this study, a phosphorimetric and colorimetric dual-signaling strategy has been exploited for the determination of OPPs for the first time. Comparing with steady-state fluorescence, room-temperature phosphorescence (RTP) possesses some distinguished charac-
INTRODUCTION Residual pesticide-induced contaminated foods have gained increasing public attention and become one of the hot global food safety issues.1,2 Organophosphorous pesticides (OPPs), a subclass of pesticides, are most extensively applied in agriculture throughout the world to boost agricultural productivity and improve the quality of agricultural products due to their high efficiency for eliminating insects, relatively low persistence under natural conditions, easy preparation, and low cost.1,2 On the basis of statistics from Pesticide Action Network North America (PANNA), the percentage of OPPs in the insecticides presently used in United States is scaled to ∼70%.3 It has been reported that these pesticides exhibit acute toxicity to humans even though their residue concentrations in contaminated agricultural products and water are very low.1,2 The toxicity of OPPs is attributed to their ability to irreversibly suppress the activity of acetylcholinesterase (AChE), a necessary enzyme in the central and peripheral nervous system to decompose the neurotransmitter acetylcholine at cholinergic synapses.4 The suppression of AChE makes acetylcholine active in the synapse and results in the accumulation of acetylcholine in the body, leading to fatal risks.4 It is greatly desired to develop reliable methods with high sensitivity for assaying pesticide residues in complex samples to guarantee food security and ecosystem safety. Different analytical techniques, such as chromatography coupled with mass spectrometry,5 enzyme-linked immunosorbent assay (ELISA),6 reflective interferometric Fourier transform spectroscopy (RIFTS),7 surface-enhanced Raman scattering (SERS) technique,8 molecular imprinting technique,9 fluori© XXXX American Chemical Society
Received: May 4, 2015 Revised: September 27, 2015 Accepted: September 27, 2015
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DOI: 10.1021/acs.jafc.5b03096 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry teristics such as longer emission lifetime and delayed relaxation time, making the lowest background noises from short-lived autofluorescence and scattering light of the analytical system to be achieved; therefore, RTP is favorable for analysis of complex samples as a signal transduction method.19−31 Recently, the RTP of doped quantum dots (QDs), e.g., Mn-doped ZnS QDs, has attracted growing attention on optosensing.20−31 Doped QDs have been considered as a novel type of luminescent material and displayed promising applications in fabricating chemosensors, biosensors, and bioimaging based on their intrinsic advantages of conventional QDs.20 However, most research has been interested in developing QD-based fluorescent chemo/biosensors; the distinguished phosphorescent properties of QDs still have not been used sufficiently for applications in analytical chemistry.20 As far as we know, such RTP of QDs used to detect OPPs has rarely been reported.25 On the other hand, colorimetric assays, which can be achieved with naked eyes or UV/vis spectrometry, have shown some advantages in their simpleness such as simple instrumentation and easy operation procedures.32−37 Typically, colorimetric detections based on gold nanoparticles (AuNPs), which generally display different colors in the visible range resulting from their localized surface plasmon resonance (LSPR) absorption and scattering,36,37 have been applied for a variety of analytes including biomacromolecules, small molecules, and metal ions.32−37 We believe that the sensing strategy combining phosphorimetric and colorimetric techniques will provide a novel method with simplicity and high sensitivity for analytical applications. In this study, we demonstrate a dual-signaling sensing system by taking advantages of both phosphorimetric and colorimetric methods for the first time, which relies on the inner filter effect (IFE) of AuNPs on the phosphorescence of Mn-ZnS QDs. In this IFE system, AuNPs and Mn-ZnS QDs are designed as the absorber−phosphor pair to regulate the phosphorescence emission of Mn-ZnS QDs. The IFE of luminescent species is generally defined as the absorption of the excitation and/or emission of lumiphores by absorbers and therefore leads to the quenching of lumiphores in the IFE systems.38 More importantly, the linking between the absorbers and the lumiphore is not required in IFE detection systems, making the IFE-based detection considerably flexible and simpler for analytical applications. On the other hand, an improved detection sensitivity may be obtained compared with the absorbance or luminescence intensity alone because the varying absorbance of absorbers results in exponential variance in the luminescence of lumiphores.38 Recently, a few studies have demonstrated IFE-based applications in developing novel fluorescence assays, although it usually introduces undesired systematic errors to fluorescence measurements and is expected to be avoided.39−45 However, to the best our knowledge, analytical application that relies on IFE of AuNPs on the phosphorescence of QDs has not been reported so far. In the present study, we describe a colorimetric and phosphorimetric dual-signaling strategy for sensitive assay of OPPs including paraoxon, parathion, omethoate, and dimethyl dichlorovinyl phosphate (DDVP) (Figure 1). The assay principle is attributed to the remarkable quenching of the phosphorescence of Mn-ZnS QDs by AuNPs via IFE and the inhibition of the activity of acetylcholinesterase (AChE) by OPPs. In the presence of OPPs, the production of thiocholine from the hydrolysis of acetylthiocholine catalyzed by AChE is hindered, resulting in the aggregation of AuNPs and thereby the
Figure 1. Chemical structures of organophosphorus pesticides tested in this study.
restoration of the IFE-quenched phosphorescence of Mn-ZnS QDs. Consequently, the aggregation of AuNPs and the phosphorescence recovery of Mn-ZnS QDs are dependent on OPP concentration. On the basis of the variations of absorbance and phosphorescence of the analytical system, a dual-readout assay for OPPs has been proposed.
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EXPERIMENTAL PROCEDURES
Materials and Apparatus. ZnSO4·7H2O, Na2S·9H2O, MnCl2· 4H2O, mercaptopropionic acid (MPA), HAuCl4·H2O, sodium citrate, acetylcholinesterase (AChE, from Electrophorus electricus), and acetylthiocholine iodide were provided by Sigma-Aldrich (Tianjing, China). Paraoxon, dimethyl dichlorovinyl phosphate (DDVP), parathion, and omethoate were obtained from Aladdin-Reagent Co. (Beijing, China). The reagents used in this study are of analytical grade and without further purification. The ultrapure water (Mill-Q, Millipore, 18.2 MΩ resistivity) was used for all the experiments in this study. Mn-Doped ZnS QDs modified with MPA and citratemodified AuNPs were synthesized with the procedures reported in the literature.30,31,46 UV/vis absorption spectra were performed on a UV-3010 spectrophotometer (Hitachi, Japan). Phosphorescence spectra were recorded on an LS-55 fluorescence spectrophotometer (PerkinElmer Company, U.S.A.) using a quartz cell (1 cm × 1 cm) in the phosphorescence mode. Decay curve measurements were carried out on a model FLS920 steady-state and lifetime spectrometer (Edinburgh Instruments Ltd., Livingston, U.K.). Apple juice was prepared with a juice extractor. The freshly prepared aqueous solution of acetylthiocholine (1.0 mM) was used immediately (not exceeding 3 h) to reduce its hydrolysis. The stock solution of pesticides (4.0 mM) was first prepared with methanol and then was diluted to desired concentrations with ultrapure water. AChE (1.0 U/mL) was prepared with phosphate-buffered solution (pH 8.0, 10 mM) and used immediately for the following experiments. Experimental Procedures for the Assay of OPPs. Paraoxon, one typical OPP frequently used, was selected as the representative pesticide for carrying out the experiments. AChE (20 μL, 1 U/mL) was first mixed with 0.5 mL of phosphate buffer containing different concentrations of paraoxon at 37 °C for 20 min. Acetylthiocholine (20 μL, 1 mM), AuNPs (0.67 mL, 12 nM), and Mn-ZnS QDs (0.2 mL, 40 mg/L) were introduced into each mixture, respectively, and then the resulting mixtures were scaled to 2.0 mL using phosphate buffer and mixed thoroughly. After incubating in the dark for 10 min, UV/vis absorption and phosphorescence spectra were recorded, respectively, and the phosphorescence emission spectra were measured at 580 nm under the excitation at 290 nm. All measurements were repeated at least five times. B
DOI: 10.1021/acs.jafc.5b03096 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Figure 2. (A) Overlay spectra of absorption spectrum of AuNPs (a) and phosphorescence emission spectrum of Mn-ZnS QDs (b), (B) phosphorescence spectra of Mn-ZnS QDs in the presence of various concentrations of AuNPs (from top to bottom: 0, 0.2, 0.6, 0.8, 1.0, 1.3, 1.5, 1.8, 2.0, 2.5, 2.8, 3.0, 3.2, 3.5, 4.0 nM), (C) absorption spectrum of AuNPs in the absence (a) and presence (b) of Mn-ZnS QDs(C), and (D) the phosphorescence lifetime of Mn-ZnS QDs in the absence (green line) and presence of 1.0 nM (red line) and 2.0 nM (black line) AuNPs. Unless otherwise stated, the concentrations for Mn-ZnS QDs and AuNPs are 4 mg/L and 4.0 nM, respectively. The excitation wavelength for phosphorescence measurements is at 290 nm. Analysis of Paraoxon in Spiked Samples and Contaminated Samples. In this study, lake water and apple juice made from clean apples (i.e., without sprayed OPPs) were used for spiked experiments, while the apple juice made from apples sprayed with OPPs was used for investigating OPP residues with time. The apples (with or without sprayed OPPs) were smashed into pieces and then squeezed into homogenates with juice squeezer. The obtained homogenates (20 g) were dissolved into 20 mL of methanol, and the resultant dispersion was filtered with a 0.22 μm filter (PES membrane) to get rid of the insoluble matrixes,12 and then apple juice was collected for further experiments. For spiked experiments, different volumes of paraoxon stock solutions (4.0 mM, i.e., 1.1 g/L) were incubated in the prepared 4.0 mL of real sample (lake water or apple juice) and then scaled to 5.0 mL using buffer, resulting in final concentrations of paraoxon of 5.0 nM, 10.0 nM, and 100.0 mM, respectively. Five μL of the resulting sample was mixed with AChE (20 μL, 1U/mL) and incubated at 37 °C for 20 min, following by the introduce of acetylthiocholine (20 μL, 1 mM), AuNPs (0.67 mL, 12 nM), and Mn-ZnS QDs (0.2 mL, 40 mg/L). The mixture was then scaled to 2.0 mL by phosphate buffer, mixed thoroughly, and incubated for 10 min in the dark, and then UV/ vis absorption and phosphorescence were recorded. For the contaminated apple juice, similar analytical procedures were adopted.
absorption coefficient, which limits the IFE efficiency and therefore reduces the sensitivity of the IFE-based analytical system. Consequently, such luminescent method based on IFE has not been extensively exploited so far.38,43 Undoubtedly, to get high sensitivity for IFE-based detection, the important thing is to improve the inner filter efficiency and, thus, to achieve a low luminescent background.38,43 To such an attempt, a practical approach is to expand the spectral overlapping of absorber absorption with lumiphore emission, and/or to select proper absorber with larger absorption coefficient.38,43 In this study, AuNPs have been chosen as the absorbers, which is based on the fact that AuNPs possess a much larger absorption coefficient (e.g., 2.7 × 108 /M/cm at 520 nm for the particles with a diameter of 13 nm46) compared to those of usual chromophores. Figure 2A shows a good overlap of the absorption band of AuNPs (curve a) with the phosphorescence emission band of Mn-ZnS QDs (curve b), which provides a precondition for possible IFE between AuNPs and Mn-ZnS QDs. To confirm the IFE occurred between AuNPs and MnZnS QDs, the phosphorescence spectra of Mn-ZnS QDs were investigated when Mn-ZnS QDs were mixed with various concentrations of AuNPs. We can see from Figure 2B that the phosphorescence emission of Mn-ZnS QDs gradually decreases upon increasing concentrations of AuNPs. In the present system (pH 8.0), the AuNPs obtained with citrate reduction method were capped with citrate and negatively charged, supported by its zeta potential, −36.6 mV, because of the ionization of the −COOH group in citrate.44 The zeta potential
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RESULTS AND DISCUSSION IFE of AuNPs on Phosphorescence of Mn-ZnS QDs. Generally, the key point in designing an IFE-based analytical system is to select a suitable absorber/lumiphore pair because the effective IFE could happen under the conditions of sufficient spectral overlapping between the absorber absorption and the excitation and/or emission of the lumiphore.38,43 However, the conventional absorber generally possesses a small C
DOI: 10.1021/acs.jafc.5b03096 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Figure 3. (A) UV/vis absorption spectra of AuNPs (a), Mn-ZnS QD−AuNP mixed solution (b), Mn-ZnS QD−AuNP solution incubated with AChE and acetylthiocholine (c), and Mn-ZnS QD−AuNP solution containing AChE was pretreated with paraoxon, and acetylthiocholine (d); (B) phosphorescence spectra of Mn-ZnS QDs (a), Mn-ZnS QD−AuNP mixed solution (b), Mn-ZnS QD−AuNP mixed solution incubated with AChE and acetylthiocholine (c), and Mn-ZnS QD−AuNP mixed solution containing AChE was pretreated with paraoxon and acetylthiocholine (d). The concentrations for AChE, acetylthiocholine, AuNPs, Mn-ZnS QDs, and paraoxon are 10 mU/mL, 10 μM, 4 nM, 4 mg/L, and 1 μM, respectively.
as that of pure AuNPs solution (curve a in Figure 3A), and they displayed the typical characteristic absorption at 520 nm. Upon the addition of both acetylthiocholine and AChE into a MnZnS QD−AuNP mixed solution, the quenched phosphorescence of Mn-ZnS QDs (curve b in Figure 3B) recovered significantly (curve c in Figure 3B), followed by the decreased of absorbance and the slight shift of the absorption band (solution color was changed from red to purple) (curve c in Figure 3A). To investigate the potential application in pesticides determination, AChE (10 mU/mL) was first mixed with paraoxon (1.0 μM) and incubated for 20 min at 37 °C; acetylthiocholine (10 μM), AuNPs (4.0 nM), and Mn-ZnS QDs (4.0 mg/L) were then mixed together in sequence. We found that the absorbance was recovered with a duration of 30 min (curve d in Figure 3A), and phosphorescence recovery of Mn-ZnS QDs was impeded (curve d in Figure 3B), accompanied by the change in the color of the mixed solution from purple to red. These results indicate that paraoxon can surpress the activity of AChE, thus hindering the production of thiolcholine from the hydrolysis of acetylthiocholine catalyzed by AChE. Consequently, the absence of paraoxon can not only lead to absorbance change (also color change) but also can lead to the phosphorescence recovery of Mn-ZnS QD−AuNP solutions. Additionally, we can clearly see the aggregating behavior of AuNPs that resulted from the addition of AChE and acetylthiocholine from the analysis of transmission electron microscopy (TEM). Upon the addition of AChE and acetylthiocholine, the monodispersed AuNPs show a tendency to aggregate together and form AuNPs aggregates, which is agreement with the spectral changes displayed in Figure 3A (curve c). However, AuNP aggregates are prevented while inhibiting AChE with paraoxon. TEM analysis is also in agreement with the transforms in the aggregation states of AuNPs, demonstrating that paraoxon has the ability to prevent and form AuNP aggregates. Analytical Performances of the AuNPs−Mn-ZnS QDs Dual-Signal Readout System for OPPs Assay. To investigate the analytical performances of the present system for the assay of OPPs, we first investigated one of the most commonly used OPPs, paraoxon, as the representative model pesticide. Prior to detection, paraoxon with different concentrations was introduced to aliquots of AChE solution, respectively, and the mixtures were incubated at 37 °C to
of the MPA-capped Mn-ZnS QDs was also measured, and the zeta potential was −46 mV in pH 8.0 phosphate buffer because of the ionization of the −COOH group in MPA (pKCOOH = 4.3247). The electrostatic repulsion interaction causes the formation of a complex, or energy transfer could not happen in the present system because the plasmon absorption band of AuNPs did not show obvious changes in the presence of MnZnS QDs (Figure 2C).43,48 Moreover, the phosphorescence lifetimes of Mn-ZnS QDs that coexisted with or without AuNPs were compared. From Figure 2D, no obvious changes in the average lifetime of the Mn-ZnS QDs were observed in the presence of AuNPs, suggesting that the complex resulting from Mn-ZnS QDs and AuNPs could not be formed through hydrogen bonding or electrostatic forces.49 The observation from the studies on phosphorescence lifetime further confirms that there existed an efficient IFE of AuNPs on the phosphorescence of Mn-ZnS QDs, while no energy transfer between Mn-ZnS QDs and AuNPs occurs because nonradiative energy transfer basically changes the excitation lifetime of the energy donor.49 On the basis of these experimental results, we believed that the decreased phosphorescence emission may be mainly ascribed to the IFE of AuNPs on the phosphorescence of Mn-ZnS QDs rather than other possible processes. Notably, the quenching efficiency (Eq) of QDs by the IFE of AuNPs can be calculated via the equation: Eq = 1 − P /P0
Herein, P and P0 are the phosphorescence intensity in the presence and absence of AuNPs, respectively. In this system, the maximum Eq was estimated to be as high as 80.2% in the presence of as low as 4.0 nM Au NPs, which is much greater than the IFE-based fluorescent analytical system.43 This high quenching efficiency can be ascribed to the large absorption coefficient of AuNPs, and such a low background of the system is highly desirable for sensitive phosphorescence assay. Dual-Signal Mechanism for OPP Assay through Inhibiting AChE Activity by OPPs. To construct a system for the OPP assay via the modulation of IFE of AuNPs on phosphorescence of Mn-ZnS QDs, a mixed solution containing optimized concentration of 4.0 nM AuNPs and 4.0 mg/L MnZnS QDs was first prepared. It can be seen that the absorbance and color of the as-prepared solution (the solution color is red) (curve b in Figure 3A) remained unchanged and were the same D
DOI: 10.1021/acs.jafc.5b03096 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Figure 4. Absorption (A) and phosphorescence (C) spectra of the present system in the presence of increasing concentrations of paraoxon, and the linear calibration of (B) A650/A520 and (D) P/P0 versus the logarithm of paraoxon concentration. The concentrations for AChE, acetylthiocholine, AuNPs, and Mn-ZnS QDs are the same as those in Figure 2.
Figure 5. Normalized (A) absorbance and (B) phosphorescence response of different OPPs with the same concentration of 200 nM toward the present system. The concentrations for AChE, acetylthiocholine, AuNPs, and Mn-ZnS QDs are the same as those in Figure 2.
in Figure 4A, upon increasing the paraoxon concentrations, the absorption peak located at 520 nm increased gradually, accompanied with the gradual decrease of absorption band ranging from 600 to 800 nm. The correlation analysis of A650/ A520 (the absorbance ratio at 650 and 520 nm) against the concentrations of the paraoxon displayed a linear relationship (Figure 4B), and the larger A650/A520 values indicated lower levels in aggregation of AuNPs, consistent with lower paraoxon concentrations. In this study, good linear relationships with different slopes ranging from 10−11 to 10−7 M (i.e., 2.75 ×
react for 20 min. AuNPs, Mn-ZnS QDs, and acetylthiocholine were then introduced into each mixture in sequence. After incubating for 10 min, it can be seen that the color of the solution transforms gradually from red to purple, which is dependent on paraoxon concentrations. The UV/vis absorption spectra displayed in Figure 4A demonstrated that the absorption band was slightly red-shifted, further confirming the color transforming arising from aggregation of AuNPs. The aggregation and relevant color transforming of AuNPs that resulted from AChE-catalysis hydrolysis could be exploited for fabricating colorimetric (visual) sensor of paraoxon. As shown E
DOI: 10.1021/acs.jafc.5b03096 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry Table 1. Results of the Determination of Paraoxon in Spiked Real Samplesa colorimetric method sample
spiked/nM
lake water
0 5.0 10.0 100.0 0 5.0 10.0 100.0
apple juice
a
RTP method
found/nM
recovery/%
RSDb/%
found/nM
recovery/%
RSDb/%
4.93 ± 0.01 10.23 ± 0.03 104.5 ± 0.6
98.6 ± 0.2 102.3 ± 0.3 104.5 ± 0.6
1.45 1.98 2.23
4.95 ± 0.01 10.09 ± 0.02 103.8 ± 0.4
99.0 ± 0.2 100.9 ± 0.2 103.8 ± 0.4
1.24 2.03 1.54
4.84 ± 0.02 10.32 ± 0.01 102.8 ± 0.7
96.8 ± 0.4 103.2 ± 0.1 102.8 ± 0.7
1.81 2.15 3.00
4.97 ± 0.02 10.12 ± 0.02 101.9 ± 0.3
98.8 ± 0.4 101.2 ± 0.2 101.9 ± 0.3
1.11 1.95 2.97
Average of five repeated measurements. bRSD represents relative standard deviation.
10−9−2.75 × 10−5 g/L) and 10−7 to 10−6 M (i.e., 2.75 × 10−5− 2.75 × 10−4 g/L) were obtained, respectively. Furthermore, on the basis of the IFE of AuNPs on Mn-ZnS QDs, a phosphorescence method for paraoxon assay was also constructed. It can be seen from Figure 4C that phosphorescence emission of Mn-ZnS QDs decreased gradually upon increasing the paraoxon concentration, corresponding to the increasing of the absorbance of AuNPs at 520 nm. As shown in Figure 4D, the intensities of phosphorescence emission of MnZnS QDs were concentration-dependent, and higher concentrations of paraoxon resulted in less phosphorescence recovery. The linear plots of the relative phosphorescence intensity (P/ P0) against paraoxon concentrations ranging from 10−11 to 10−7 M (i.e., 2.75 × 10−9−2.75 × 10−5 g/L) and 10−7 to 10−6 M (i.e., 2.75 × 10−5−2.75 × 10−4 g/L) (Figure 4D) were obtained, respectively. The detection limit (3σ/s) for paraoxon (lower concentration range) was found to be 1.07 pM (0.29 ng/L). This lowest detectable concentration is much lower than the maximum residue limits (MRL) in European Union pesticides database (0.01 mg/L).50 The low detection limit is also much lower than that of previous assay of paraoxon.11,13 The repeatability of the present assay was also investigated by measuring 50 nM paraoxon, and the relative standard deviation (RSD) for seven repeated detection was calculated to be 3.73%, suggesting that the response of the present dual-signal system to paraoxon is highly reproducible. The present dual-readout strategy for the assay of other three usual OPPs including DDVP, parathion, and omethoate was also investigated using the same detection procedures designed for paraoxon. The results show that the inhibition responses of these three OPPs are still dependent on the concentration of pesticides. The inhibition responses of the typical OPPs at the concentration of 200 nM to the system were compared. From the UV/vis absorption and corresponding phosphorescence response, the inhibition efficiency could be evaluated. As shown in Figure 5, the inhibition efficiencies of the four OPPs are different and display an order (from strong to weak) of paraoxon, DDVP, parathion, and omethoate. These results also suggested that the established biosensors could be used for determination of different kinds of OPPs. The limits of detection (3σ/s) for parathion, omethoate, and DDVP are found to be 2.03 pM (0.59 ng/L), 3.12 pM (0.67 ng/L), and 1.98 pM (0.44 ng/L), respectively. These lowest detectable concentrations are also much lower than MRLs that originated from the European Union pesticides database (MRLs are 0.05 mg/L and 0.02 mg/L for parathion andomethoate, and and not shown for DDVP).50 Anti-Interference Capability of the Present System for OPPs. For demonstrating the specificity of the present system
for OPP assay, the influences of potential different interfering substances including glucose, sucrose and sodium acetate, histidine, tryptophan, and glycerol were investigated under the same experimental conditions. We choose paraoxon detection as a model, and the selectivity of phosphorescence for paraoxon detection was evaluated by testing a mixture of paraoxon with different interfering substances. The results demonstrate that the phosphorescence responses changed less when the interfering species with identical concentration were present in the present system, indicating that the present system displays high selectivity for assaying paraoxon and, therefore, can be applied for OPP detection in real samples. Detection of OPP Residues in Spiked Samples and Authentic Contaminated Samples. To demonstrate the real applications of the present method for selective assaying of OPPs, we chose paraoxon as the representative model pesticide and the amounts of paraoxon in local natural lake water and apple juice samples were detected using the standard addition method. Paraoxon with different concentrations including 5.0, 10.0, and 100.0 nM was introduced into lake water and apple juice solutions, respectively, and then detected by the proposed colorimetric and phosphorimetric method. The determinated results are displayed in Table 1, and it can be seen that excellent recoveries ranging from 96.8 ± 0.4% to 104.5 ± 0.6% were obtained and the RSDs were
