Article pubs.acs.org/ac

Conversion of Inhibition Biosensing to Substrate-Like Biosensing for Quinalphos Selective Detection Limin Yang, Juan Han, Wei Liu, Jiqiang Li, and Lei Jiang* State Key Laboratory of Heavy Oil Processing and Center for Bioengineering and Biotechnology, China University of Petroleum (East China), Qingdao, Shandong 266555, P. R. China S Supporting Information *

ABSTRACT: Since all of the organophosphorus pesticides (OPP) inhibit the cholinesterases with a common mechanism, it is still challenging to detect OPP selectively with inhibition-based biosensors. This study focuses on the conversion of a typical inhibition biosensing to a selective substrate-like biosensing. The interaction of quinalphos with plant-esterase involves not only a decrease in enzyme activity but also a heterolytic bond cleavage of quinalphos. The leaving group eliminated from quinalphos is an ideal biomarker due to its specificity in most OPP. Thus, using 2hydroxyquinoxaline (HQO), the leaving group of quinalphos, as the biomarker and meso-tetra (4-sulfonatophenyl) porphine (TPPS4) as an optical probe, quinalphos can be selectively detected. The molecular recognition between TPPS4 and HQO leads to a considerable sensitivity of the detection. The spectral responses of TPPS4 show a linear dependence on quinalphos concentration in the presence of plant-esterase within the 0.01−1 mg kg−1 range. The detection limit is 0.01 mg kg−1, well below the maximum residue limits (MRLs) defined by European Union (0.05 mg kg−1) and China (0.2 mg kg−1).

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measurement of inhibition also provides the analytical basis for their sensing. Enzyme inhibition biosensors have emerged as the most promising alternative to chromatographic methods for faster and simpler detection of OPP since 1962,24 in which the enzymatic activity is employed as an indicator of pesticides quantity. Acetylcholinesterase (AChE, EC 3.1.1.7) has been the most used bioreceptor for such biosensors. Various inhibition biosensor systems, based on the combination of AChE with amperometric,25−28 potentiometric,29 optical,30−33 or piezoelectric transducers,34 have been proposed for field screening of OPP. Their simplicity, high sensitivity, and low cost make in situ measurement of OPP possible. To improve the response and stability in trace pesticide detection, nanomaterials like graphene,35,36 carbon nanotubes,37,38 metal nanoparticles,39−41 and quantum dots42−45 have also been introduced recently. These nano matrices are used as signal transducers to mediate current flow or as recognition agents and electroactive tags to detect the analytes.46,47 The stability, sensitivity, and detection limit in OPP determination in the pico- to nanomolar concentration range have been significantly promoted. Despite many efforts to develop inhibition biosensors, some analytical characteristics of these systems hinder their transformation into practical analytical tools. The major problem is the lack of selectivity.48−50 The quantification of anticholinesterase pesticides is based on the measurement of the decreased enzyme activity after exposure. Since inhibition is caused by a

ood safety issues caused by pesticide residues have become the spotlight of public concerns all over the world.1 Oranophosphorus pesticides (OPP), esters of phosphorusbased acid derivatives, account for over 38% of the total pesticides used worldwide due to their low cost, broadspectrum insecticidal activity, and relatively low persistence in the environment.2,3 The use of OPP provides benefits for increasing agricultural production. However, their extensive use also has given rise to the risk of OPP residues in food and drinking water and poses a high threat to public health.4 According to the World Health Organization (WHO), about three million acute and severe OPP poisonings occur annually, with more than 220 000 fatalities.5 A leading priority of human health security is to develop sensitive, selective, and reliable analytical methods for determination of OPP residues. Although the current reference methods are primarily chromatographic, such as gas chromatography (GC),6−8 gas chromatography−mass spectrometry (GC/MS),9,10 liquid chromatography−mass spectrometry (LC-MS),11−13 high-performance liquid chromatography (HPLC),14,15 capillary gas chromatography (CGC),16 and gel permeation chromatography-gas chromatography (GPC-GC),17−19 the application of these methods are limited due to the requirement of expensive equipment, tedious sample pretreatment, and a drawn-out assay procedure. A promising alternative involves the use of enzymelinked immunoassay (ELISA).20−23 This technique has often demonstrated the required sensitivity and acceptable specificity; however, it needs specific antibodies and, indirectly, the use of animals in order to produce “receptors”. Since the toxicity of OPP is commonly attributed to their irreversible inhibitory effects on the cholinesterases, the © XXXX American Chemical Society

Received: January 29, 2015 Accepted: April 29, 2015

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Determination of Kinetic Constants for the Interaction of Quinalphos with Plant-Esterase. The kinetic parameters for the interaction of quinalphos with plant-esterase were determined spectrophotometrically in the presence of substrate (1-naphthyl acetate, 1-NA) and chromogen (Fast Blue B) as described previously.64 A substrate concentration of 0.08 mM and chromogen concentration of 0.0067% (w/v) were chosen for all kinetic experiments. PBS buffer (0.1 M, pH 7.0) was used as the supporting solution. Briefly, diluted quinalphos was added to the mixture of 1-NA and Fast Blue B to give final concentrations of 1−6.67 mg kg−1. The reaction of 1-NA hydrolysis was started by adding plant-esterase (final concentration, 4 μg mL−1) to the sample, while buffer only was added to the reference. Both were immediately transferred to the thermostated compartment of the spectrophotometer (UV-2450, Shimadzu, Japan) at 30 °C. Product formation was continuously monitored via the increase of optical density at 535 nm. By using a reference, a correction was made directly for nonenzymatic hydrolysis of the substrate. Preparation of Quinalphos Modified Plant-Esterase. The quinalphos modified plant-esterase was prepared by the incubation of plant-esterase with quinalphos in PBS buffer (0.1 M, pH 7.0). Briefly, plant-esterase (final concentration, 0.08 mg mL−1) with quinalphos (final concentration, 2.5 g kg−1) was mixed and incubated for 5 h at 4 °C. The decrease in enzyme activity was monitored until inhibition was complete (see the Supporting Information Figure S-1). The resulting solution was first purified with a PD-10 column (GE) to remove unbound quinalphos, solvent acetone, and the HQO. Then, the resultant modified plant-esterase was concentrated by ultrafiltration using a centrifugal filter (10 kDa cutoff, Amicon Ultra-15, Millipore) by centrifuging at 10 000g for 10 min, and finally it was stored at −20 °C for future use. The protein concentration of the stock solution was determined to be ∼0.8 mg mL−1 by the Bradford dye-binding assay.65 Characterization of Quinalphos Modified Plant-Esterase. Fourier Transform Infrared (FT-IR) Spectroscopy. Infrared spectra were collected using a Nicolet 6700 FT-IR spectrometer (Thermo Scientific, USA). Control plant-esterase and quinalphos modified plant-esterase were dried under vacuum freeze-drying. The dry protein samples obtained (approximately 0.5 mg) were mixed and ground with 300 mg of potassium bromide and then compressed to transparent tablets. After collecting the background spectrum, the spectra of the tablet samples were recorded by averaging 256 scans over the frequency range of 4000 to 400 cm−1 with a spectral resolution of 4 cm−1. Second derivative spectra (Savitsky-Golay derivative) were obtained using the Ominic software to determine the component peak positions. Liquid Chromatography-Tandem Mass Spectrometry (LCMS/MS). Prior to the MS characterization, control and quinalphos modified plant-esterase were precipitated with icecold acetone (1:4 v/v) first. After acetone had been decanted, the remaining pellet was dried under vacuum. The dried pellet was resolubilized in ammonium bicarbonate buffer (0.025M, pH 8.0) and treated with trypsin at a protein ratio of 1:50 at 37 °C overnight. Proteolysis was stopped by flash freezing at −80 °C. The tryptic peptides were then lyophilized and dissolved in acetonitrile containing 0.1% formic acid for the following MS analysis. LC-MS/MS analyses of the plant-esterase peptides were performed using an LTQ XL linear ion trap mass spectrometer

range of toxic compounds (all organophosphorus and carbamate pesticides,51 heavy metals,52 aflatoxins,53 etc.), proper differentiation and identification of either an individual or a class of pesticides is difficult. In addition, the detection of inhibitors involves at least two steps: the measurement of the initial response of the sensor and its decay after contact with the inhibitor solution. This requires the addition or changing of several reagents except the enzyme.50 This makes the measurement of inhibition complicated and difficult to operate. These biosensors are thus suitable only as a screening to provide a rapid response and signal for the existence of contaminated samples. There is a need to meet the advancement of new analytical methods. All OPP potentially have a common mechanism of toxicity, the phosphorylation of AChE causing accumulation of acetylcholine, overstimulation of cholinergic receptors, and consequent clinical signs of cholinergic toxicity.54,55 This phosphorylation occurs by the nucleophilic attack of the phosphorus of OPP by the oxygen of serine (Ser-203) in the enzyme active site.56−58 Accompanied by the heterolytic cleavage of the carbon−phosphorus bond, two products, the phosphorylated enzyme and the leaving group of OPP, are obtained.59 In this respect, OPP is like a substrate. The enzyme metabolizes OPP into detectable compounds, making the conversion of inhibition biosensors to a substrate-like biosensor possible. In such a biosensor, the selectivity is provided by the product of the enzyme reaction. In this paper, we report an unusual yet highly selective substrate-like biosensing method for the detection of quinalphos (O,O-diethyl-O-quinoxalin-2-yl-phosphoro-thioate), an important OPP effective against a great variety of biting and sucking pests on cotton, vegetables, and fruits. An esterase (i.e., plant-esterase, EC 3.1.1.X) extracted from plants was used as the bioreceptor alternative to AChE due to its low cost, easy extraction, convenient preservation, and high sensitivity comparable with cholinesterase.60,61 A water-soluble porphyrin, meso-tetra (4-sulfonatophenyl) porphine (TPPS4), is introduced as the in situ probe for the metabolites of quinalphos. Upon binding to the biomarker, porphyrins experience significant variations of their optical properties, making them potentially sensitive within sensors.62



EXPERIMENTAL SECTION Reagents and Materials. Plant-esterase was extracted from wheat flour and further purified to homogeneity as described previously.63 The enzyme preparation was stored frozen at −20 °C in small aliquots. TPPS4 was obtained as tetrasodium salt from Sigma-Aldrich (USA). The stock solutions of TPPS4 (1 mM) were prepared in phosphate buffered saline (PBS, 0.1 M, pH 7.0) before use. The analytical standards of OPP were purchased from Dr. Ehrenstorfer GmbH (Germany), including quinalphos, parathion, chlorpyrifos, fenitrothion, phosmet, isocarbophos, phorate, dichlovos, acephate, methamidophos, dimethoate, and omethoate. The stock solutions of each OPP were prepared at 50 g kg−1 in acetone, stored in darkness at 4 °C, and appropriately diluted in PBS buffer (0.1 M, pH 7.0) just before the experiment so that the acetone concentration within any incubation never exceeded 5% and had no significant effect on plant-esterase activity. 2-Hydroxyquinoxaline (HQO) was obtained from Sigma-Aldrich (USA). Other chemicals used were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All compounds were used without further purification. B

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To confirm the selectivity derives from the combined action of plant-esterase and TPPS4, the responses of the assay on quinalphos and other OPP were also investigated in the absence of plant-esterase. Briefly, each OPP was appropriately diluted with PBS buffer and then directly mixed with TPPS4 solution. After incubation in darkness overnight, the absorbance spectra were collected. All experiments were performed in triplicate.

(Thermo Fisher, USA) coupled with a Surveyor Plus HPLC system. Twenty μL of peptide solution was loaded into the CTICAP5150100 reverse phase capillary column (Column Technology Inc. USA). The column temperature was maintained at 25 °C, and flow rate was 200 μL min−1. Mobile phase A contained 0.1% formic acid in water, and B contained 0.1% formic acid in acetonitrile. After initial loading at 5% B over 15 min, the gradient increased to 32% B over 45 min then ramped to 90% B over 35 min. To wash the column, the 5% B was maintained for 5 min, while the equilibration step was carried out for an additional 20 min. Peptides were ionized using nanoelectrospray in the positive mode and automatically detected in MS/MS mode from 400 to 1800 m/z. The collision energy was set at 35%. SEQUEST was used for searching the Triticum aestivum database for peptide sequence and identification of the diethyl phosphorothionate (which results from the formation of quinalphos-plant-esterase adduct) with neutral scan loss of 170.0167 Da (monoisotopic mass) or monoethyl phosphorothionate (which results from “aging” of the adduct) with a neutral scan loss of 141.9854 Da (monoisotopic mass). Detection of Quinalphos. Quinalphos inhibits the activity of the plant-esterase and induces the formation of a new absorption band in the spectrum of TPPS4. In the present study, the response to quinalphos was calculated by dividing the absorbance of the TPPS4 solution at 432 nm by the absorbance at 414 nm (A432 nm/A414 nm). In a typical experiment, the proper amount of quinalphos stock was at first diluted with PBS buffer (0.1 M, pH 7.0) and mixed with plant-esterase (final concentration is 0.69 mg mL−1). Immediately, TPPS4 solution (final concentration is 2 μM) was added. The resulting mixtures were kept in darkness overnight at room temperature. The final quinalphos concentrations were in the range of 0.005−20 mg kg−1. Finally, the absorbance spectra were collected on a UV2450 spectrophotometer (Shimadzu, Kyoto, Japan). Wavelength scans were recorded from 350 to 750 nm using a wavelength step size of 1.0 nm. All experiments were performed in triplicate. The limit of detection (LOD) has been defined as the concentration of the species being measured which gives a minimum detectable difference signal that is equal to 2 or 3 standard deviations (S.D.) of the mean response of the blank samples. Any concentration above the LOD value has a 95% (2 S.D.) or 99% (3 S.D.) probability of being a true positive result.66 Herein, the LOD values were calculated using the following formula:



RESULTS AND DISCUSSION Metabolism of Quinalphos by Plant-Esterase. Interaction between Quinalphos and Plant-Esterase. Since the recognition of the formation of a reversible enzyme−inhibitor complex, it is widely accepted that the interaction between OPP and serine hydrolases is described by eq 1.67−69In this equation,

E−OH represents AChE and related serine hydrolases in which the serine hydroxyl moiety (−OH) is emphasized, R and R′ are a variety of different groups (alkoxy, alkyl, amino, thioalkyl, etc.), X is the leaving group, Kd is the dissociation constant between the enzyme−OPP complex and reactants, and kp is the phosphorylation constant. Plant-esterase (EC 3.1.1.X) is a carboxylic ester hydrolase similar to AChE (EC 3.1.1.7); however, the interaction mechanism of OPP with plant-esterase has not yet been defined due to the unresolved protein structure. Assuming their interaction took place via a transphosphorylation reaction similar to that between OPP and AChE, it can be shown as eq 2: Kd

kp

E + PX ⇌ [E· PX] → EP + X

(2)

This two-step process includes the reversible formation of an intermediate complex (E·PX) followed by formation of the enzyme-phosphorus bond (EP) with displacement of the leaving group (X). The reaction may be described by the dissociation constant (Kd) and the phosphorylation constant (kp). To obtain these kinetic constants, the interaction of quinalphos with plant-esterase was investigated using the recording spectrophotometric method. The progressive substrate hydrolysis in the presence of quinalphos is illustrated in Figure 1. It is observed that the quinalphos-plant-esterase interaction could be completed in 1800 s. As quinalphos concentration increases, both initial velocity and maximum velocity decrease. It indicates that quinalphos blocks the catalysis function of plant-esterase, causing the accumulation of substrates. This is consistent with the fact that OPP can inactivate plant-esterase in vitro.61 Using the combination of nonlinear regression analysis70 and the double-reciprocal method,71 the kinetic constants of Kd and kp were determined to be 2.28 × 10−4 M and 0.15 s−1, respectively (see the Supporting Information Figure S-4). Since Kd provides a measure of the dissociation of the enzyme-quinalphos intermediate complex, it is suggested that plant-esterase has similar affinity for quinalphos with that of AChE-paraoxon (Kd = 1.7 × 10−4 M).72 Once the reversible intermediate complex is formed, phosphorylation of plant-esterase may proceed rapidly and irreversibly. The irreversibility of the quinalphos-plantesterase interaction was confirmed by repeated ultrafiltration

limit of detectable response = average response of the blank + (3 × standard deviation of the blank)

Selectivity. The selectivity was investigated using other OPP (i.e., parathion, chlorpyrifos, fenitrothion, phosmet, isocarbophos, phorate, dichlovos, acephate, methamidophos, dimethoate, and omethoate). All these OPP can efficiently inhibit the activity of plant-esterase (see the Supporting Information Figure S-2). The assay was carried out following the same procedure as that for quinalphos. In brief, the proper amount of each OPP stock was diluted with PBS buffer (0.1 M, pH 7.0) and then mixed with plant-esterase. Immediately, TPPS4 solution was added. The final concentration of each OPP was 20 mg mL−1. The resulting mixtures were kept in darkness overnight at room temperature. Finally, the absorbance spectra were collected from 350 to 750 nm with a wavelength step size of 1.0 nm. C

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Figure 1. Progressive hydrolysis curves of substrate catalyzed by plantesterase with different quinalphos concentrations.

and a kinetic graph (EV plot)73 (Supporting Information Figure S-5). The resultant products are thus closely related to quinalphos quantity. Identification of Interaction Products. To identify the products from the irreversible interaction of quinalphos with plant-esterase, quinalphos modified plant-esterase was characterized with Fourier transform infrared (FT-IR) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). As shown in Figure 2a, strong transmittance bands from the P− O−C (aliphatic) linkage (1023 cm−1) and PS group (766 cm−1)74 appear in the infrared spectra of quinalphos and quinalphos modified plant-esterase but are absent in that of control plant-esterase. Furthermore, several bands associated with the quinoxalinyl group of quinalphos disappear in the spectrum of quinalphos modified plant-esterase: P−O−C (aromatic) band at 1202 cm−1, ring-stretching bands in the region of 1620−1350 cm−1, and other C−H bending and ringbreathing bands between 1300 and 1000 cm−1.74,75 These results suggest the loss of quinoxaline-2-oxyl and the formation of covalent diethyl phosphorothioic ester bond. The MS/MS spectrum (Figure 2b) also shows the confident identification of phosphorylated peptide LTLDSLYDDGTYR with the site S modified with 170.02 Da corresponding to the mass of diethyl phosphorothionate in quinalphos modified plant-esterase (Supporting Information Table S-1). No quinalphos adducts were detected on any residues for the control sample. Therefore, it is likely that two products, 2-hydroxyquinoxaline (HQO) and phosphorylated plant-esterase, are formed after quinalphos binding to plant-esterase. This is a key point for quinalphos detection with the substrate-like biosensor. Probe the Biomarker with TPPS4. Identification of the Biomarker. On the basis of the interaction between quinalphos and plant-esterase, there are several potential biomarkers, including phosphorylated plant-esterase, 2-hydroxyquinoxaline (HQO), and free quinalphos, in the system. Figure 3a represents the reaction paths of quinalphos in plant-esterase solution. In order to identify the biomarker for quinalphos detection, the spectral behavior of TPPS4 in the presence of these potential biomarkers was investigated in PBS buffer (0.1 M, pH 7.0) using UV−vis absorption spectra, respectively. As shown in Figure 3b, the electronic absorption spectrum of TPPS4 alone demonstrates an intense Soret band in the high energy region

Figure 2. (a) Typical FT-IR spectra of quinalphos (black line), control plant-esterase (blue line), and quinalphos modified plant-esterase (red line). (b) MS/MS spectrum for doubly charged peptide ions at m/z 1703.78. Product ions corresponding to sequential loss of amino acid residues from the C or N terminus are labeled as b- or y-type ions, respectively.

at 414 nm arising from the transition of a1u(π)-eg*(π) and four weak Q bands at 515, 553, 580, and 633 nm corresponding to the a2u(π)-eg*(π).76 Since the characteristic spectral features of porphyrins are dictated by the transitions of these π electrons between two higher occupied and two lower unoccupied orbitals, binding or interaction with another molecule is expected to alter the spectrophotometric characteristics of porphyrins.77,78 First, porphyrin can interact with protein via electrostatic and hydrophobic interactions.79 In neutral pH, where plant-esterase is negatively charged (pI = 4.3−4.6)63 and TPPS4 is no longer a zwitterion but rather an anion (pKa = 4.8),80 hydrophobic interactions are the dominant force.81 However, no significant variations to peak position or shape were observed after the addition of either plant-esterase or phosphorylated plantesterase except a decrease in absorbance at 414 nm (Figure 3b), probably due to a weak interaction. Quinalphos is a heterocyclic aromatic organic molecule which consists of the fusion of benzene and pyrazine. TPPS4 was expected to interact with quinalphos through π−π interaction, to introduce a change in the TPPS4 spectrum. However, after exposure to quinalphos D

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at the periphery of the porphyrin were increased, causing a bathochromic shift in the absorbance band.84 HQO can therefore be used for quinalphos detection. Probe the Biomarker with TPPS4 in Detection Experiments. In detection experiments, plant-esterase and quinalphos were mixed together first, and then, TPPS4 was added. As expected, the spectral characteristics of TPPS4 were greatly changed. The absorbance at 414 nm significantly decreased, and a second peak at 432 nm appeared with a much stronger intensity (Figure 3b). To assign these absorption peaks, a kinetic study was performed. Figure 4 shows the measured time-dependent

Figure 4. Time-dependent absorption spectra of TPPS4 and the corresponding absorbance intensity-reaction time curves (inset). [TPPS4] = 2 μM, [plant-esterase] = 0.69 mg mL−1, and [quinalphos] = 20 mg kg−1.

absorption spectra of TPPS4 and the corresponding variations of absorbance intensity with reaction time. Upon the initiation of the reaction, an intense absorption band at 414 nm can be observed. However, no absorption peak centered at 432 nm, corresponding to the formation of the final TPPS4−HQO complex, is observed in the first 42 min. This might be because the affinity of quinalphos to plant-esterase is relatively low. As the reaction proceeds, the peak at 414 nm decreases while the peak at 432 nm increases gradually, with a clear isosbestic point at 423 nm, suggesting the conversion of TPPS4 monomers to the new complex of TPPS4−HQO. The process completes in ∼10 h. To eliminate the possible interfering effect of spontaneous hydrolysis of quinalphos, a sample without plant-esterase was investigated kinetically as a control (Supporting Information Figure S-7). The absorption spectra of TPPS4 remain almost unchanged for more than 10 h. This indicates that quinalphos is relatively stable for hours in the detection limit, which is in agreement with previous literature.82 Therefore, the biomarker, produced from the interaction of quinalphos with plant-esterase, can be probed qualitatively via TPPS4. Possible Detection Mechanism. Using the data of absorption bands, we propose the detection mechanism as a three-step process:

Figure 3. (a) Reaction paths for the potential biomarkers in the quinalphos-plant-esterase system. (b) UV−vis absorption spectra of the TPPS4 before and after exposure to these biomarkers, and the spectrum obtained in quinalphos detection with plant-esterase and TPPS4. Each of the samples was incubated in a water bath at 30 °C overnight before analysis. [TPPS4] = 2 μM, [plant-esterase] = 0.69 mg mL−1, [phosphorylated plant-esterase] = 0.69 mg mL−1, [quinalphos] = 20 mg kg−1, and [HQO] = 20 mg kg−1.

(Figure 3b), the spectral change of TPPS4 was too little to detect in the measurement. This is probably due to the electrostatic anionic repulsion between the sulfonic group of TPPS4 and the thiophosphoryl group of quinalphos. On the contrary, the other product-HQO, formed by nucleophilic attack at the phosphorus atom followed by cleavage of the aromatic bond, showed much higher potential to be a biomarker. It is mainly from the hydrolysis and metabolite of quinalphos.82,83 Upon addition of HQO, the Soret absorbance decreased and a shoulder peak at ∼432 nm appeared. Unlike quinalphos, the hydroxyl group of HQO is crucial for eliminating the electrostatic repulsion forces and facilitating the formation of the resultant complex. The π-electron orbitals E

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plant‐esterase + quinalphos + TPPS4 ⎯⎯⎯⎯⎯⎯⇀ [plant‐esterase ·quinalphos] ↽⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ (1)

Transphosphorylation

+ TPPS4 ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→ phosphorylated plant‐esterase + HQO (2)

Probing

+ TPPS4 ⎯⎯⎯⎯⎯⎯⇀ phosphorylated plant‐esterase + [TPPS4 ·HQO] ↽⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ (3)

(3)

The first binding step, involving a nucleophilic attack of a serine of plant-esterase to the phosphorus atom in quinalphos, could be the rate-limiting step. The Michaelis complex, [plantesterase·quinalphos], is formed. Second, the biomarker HQO is released, and the phosphoryl group is transferred to the serine hydroxyl via the formation of a covalent diethyl phosphorothioic ester bond. Third, HQO is probed by TPPS4, resulting in the formation of a new complex (TPPS4−HQO). The absorbance bands around 414 and 432 nm represent TPPS4 monomers and the TPPS4−HQO complex, respectively. On the basis of the changes of these bands, quinalphos detection may be explained by this mechanism. Detection of Quinalphos with the Substrate-Like Biosensing. Sensitivity. Having established the substrate-like biosensing method to detect quianlphos, we further optimized the plant-esterase/TPPS4 concentration ratio in the assay to enhance the detection sensitivity. As shown in Figure S-8, Supporting Information, the A432 nm/A414 nm value increased sharply as the concentration ratio increased in the range of 0.5− 5 and reached a maximum value of 0.76 ± 0.02. This suggests that, the higher the plant-esterase concentration, the more HQO formed. However, a higher concentration of plantesterase appears to destabilize the TPPS4−HQO complex. Further increasing the ratio up to 6.25 appears to result in conversion of the TPPS4−HQO back to the monomeric form of TPPS4. The A432 nm/A414 nm value decreased. Therefore, the optimal plant-esterase/TPPS4 concentration ratio is 5. To determine the sensitivity of this assay for quinalphos, a series of detection experiments was conducted over a quinalphos concentration range of 0.005−20 mg kg−1. As the concentration of quinalphos increases, the absorption intensity at 432 nm gradually increases and that of the 414 nm peak decreases (Figure 5a), suggesting the quinalphos concentration dependent formation of the TPPS4−HQO complex. Figure 5b shows the concentration-dependent response of the assay. The signal values increase with quinalphos concentrations. Especially, there is a sharp increase when the quianlphos concentration increases above 12.5 mg kg−1. The changes in signal decrease steadily to the plateau above 17.5 mg kg−1. Using TPPS4 as a probe, quinalphos concentrations from 0.01 to 20 mg kg−1 could be readily detected with a detection limit of 0.01 mg kg−1, which is well below the maximum residue limits (MRLs) admitted by the European Union (EU) (0.05 mg kg−1)85 or China (0.2 mg kg−1).86 Moreover, the response of the assay is highly linear, with a linear regression correlation coefficient of 0.9966 at the quinalphos concentration range of 0.01−1 mg kg−1 (Figure 5b inset). Selectivity. To determine the selectivity of the assay against quinalphos, 11 other OPP (parathion, chlorpyrifos, fenitrothion, phosmet, isocarbophos, phorate, dichlovos, acephate, methamidophos, dimethoate, and omethoate) as controls were tested under the same condition. Figure 6 shows the response of the assay against these OPP in the presence or absence of plant-esterase. In the absence of plant-esterase, none of the OPP significantly changed the A432 nm/A414 nm value of the

Figure 5. Sensitivity of quinalphos detection. (a) The absorption spectra of TPPS4-plant-esterase mixtures in the absence and presence of different concentrations of quinalphos. (b) Response (A432 nm/ A414 nm) of the assay against quinalphos concentrations. Inset shows the response linearity of the assay.

Figure 6. Response of the assay against quinalphos (20 mg kg−1) and 11 other OPP (20 mg kg−1) in the presence or absence of plantesterase. [TPPS4] = 2 μM, [plant-esterase] = 0.69 mg mL−1.

F

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Article

Analytical Chemistry

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TPPS4 solutions. Instead, after plant-esterase addition, quinalphos led to a large increase in the A432 nm/A414 nm value, as expected. Therefore, the substrate-like biosensing method shows considerable sensitivity and selectivity toward quianlphos detection.



CONCLUSIONS Almost all the inhibition biosensors for OPP detection are based on inhibition after exposure, which leads to a lack of selectivity. However, inhibition actually is an irreversible process best described as a transphosphorylation reaction. The inhibition is common to a wide range of inhibitors, but the metabolites, especially the leaving group, can become the “fingerprint” for most OPP. The use of OPP like a substrate gives an opportunity to improve the selectivity of the inhibition biosensor. In this study, plant-esterase, an alternative to AChE, is used as bioreceptor. Quianlphos can be metabolized by plantesterase via a heterolytic cleavage of the carbon−phosphorus bond. Two interaction products, the phosphorylated enzyme and HQO, are obtained on the basis of FT-IR and LC-MS/MS analysis. Using HQO as the biomarker and TPPS4 as the probe, concentrations as low as 0.01 mg kg−1 can be detected, which is well below the MRL admitted by the European Union (EU) (0.05 mg kg−1) or China (0.2 mg kg−1). The assay response is highly linear in a wide concentration range (0.01−1 mg kg−1). More importantly, selectivity of the sensor was demonstrated in the presence of several other OPP at high concentrations (20 mg kg−1).



ASSOCIATED CONTENT

S Supporting Information *

Time course of enzyme activity during plant-esterase modification, detailed kinetics and reversibility of the quinalphos-plant-esterase interaction, LC-MS/MS characterization, and possible interfering effect and optimization of the substrate-like biosensing method. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b00376.



AUTHOR INFORMATION

Corresponding Author

*Telephone: +86-053286981568. E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank Dr. Robert K. Thomas from Oxford University for taking time from his busy schedule to revise our manuscript. This work was financially supported by the National Natural Science Foundation of China (31301471, 21204102), International Science & Technology Cooperation Program of China (2015DFG3660), National 863 High-Tech Project (2015AA020947), Science and Technology Development Project of Shandong Province (2014GHY115020), and the Project of Science and Technology Program for Basic Research of Qingdao (13-1-4-236-jch).



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DOI: 10.1021/acs.analchem.5b00376 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

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DOI: 10.1021/acs.analchem.5b00376 Anal. Chem. XXXX, XXX, XXX−XXX

Conversion of inhibition biosensing to substrate-like biosensing for quinalphos selective detection.

Since all of the organophosphorus pesticides (OPP) inhibit the cholinesterases with a common mechanism, it is still challenging to detect OPP selectiv...
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