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Label-Free Homogeneous Electroanalytical Platform for Pesticide Detection Based on Acetylcholinesterase-Mediated DNA Conformational Switch Integrated with Rolling Circle Amplification Xiaojuan Liu, Mengmeng Song, Ting Hou, and Feng Li ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00081 • Publication Date (Web): 30 Mar 2017 Downloaded from http://pubs.acs.org on April 2, 2017
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Label-Free Homogeneous Electroanalytical Platform for Pesticide Detection Based on Acetylcholinesterase-Mediated DNA Conformational Switch Integrated with Rolling Circle Amplification Xiaojuan Liu, Mengmeng Song, Ting Hou, and Feng Li* College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao 266109, People’s Republic of China. KEYWORDS: homogeneous electroanalysis, pesticides assay, rolling circle amplification, DNA conformational switch, label-free strategy
ABSTRACT: This study addresses the need for sensitive pesticide assay by reporting a new label-free and immobilizationfree homogeneous electroanalytical strategy, which combines acetylcholinesterase (AChE)-catalyzed hydrolysis productmediated DNA conformational switch and rolling circle amplification (RCA) to detect organophosphorous and carbamate pesticide in a “signal-on” mode. When target pesticides were present, AChE activity was inhibited and couldn’t trigger the following DNA conformational change and the RCA reaction, which results in numerous methylene blue (MB) molecules in free state, generating a strong electrochemical response. This proposed strategy was highly sensitive for omethoate detection with a detection limit as low as 2.1 μg/L and a linear range from 10 to 10000 μg/L. Furthermore, this strategy was demonstrated to be applicable for pesticide detection in real samples. Thus, this novel label-free homogenous electroanalytical strategy holds great promise for pesticide detection and can be further exploited for sensing applications in environment and food safety field.
Organophosphorous and carbamate pesticides such as omethoate, aldicarb etc. have been extensively used in agriculture across the world to obtain higher yields as they can protect crops and plants from pests and insects.1 However, irrational usage of pesticides often results in excessive residues that can lead adverse effects to environment and human health due to their high toxicity.2 This toxicity results from their strong inhibition effect on the activity of acetylcholinesterase (AChE) enzyme contained mainly in cholinergic neurons. Since AChE can catalyze the hydrolysis rate of acetylcholine into choline and acetate, the inhibition of AChE activity often causes excessive acetylcholine accretion in the body, which can affect the physiology of the nervous system and lead to fatal consequences.3 For the sake of environmental protection and public health, it is highly significant to develop sensitive strategies for reliable quantification of trace level pesticide residues in agricultural products and environmental samples. In the past decade, much effort has been made to develop efficient approaches to pesticides assay, such as gas chromatography (GC),4 gas chromatography with mass spectrometry (GC-MS),5 high performance liquid chromatography (HPLC)6 etc. Although these traditional
chromatographic methods are accurate and routinely employed for pesticides assay, they usually require preconcentration/separation steps, long analysis time, sophisticated instruments, and skilled manpower, which restrict their applications in regular environmental and food safety monitoring. To overcome these problems, various alternative approaches have been developed and utilized for the determination of pesticides, such as colorimetric approaches,7 fluorometric methods,8-10 immunoassays,11 surface plasmon resonance,12 13 electrochemiluminescence, electrochemical approaches14-16 and so on. Among them, enzyme-based electroanalytical approaches have emerged as the most attractive method for pesticides assay owing to their merits of user-friendly operation, low cost, high sensitivity, and compatibility with micro-manufacturing technology. In recent years, Enzyme inhibition-based electroanalytical methods, especially for those constructed by immobilization of AChE enzyme onto different electrode surfaces, have been widely explored and exhibited satisfactory results for pesticides detection.17-19 For instance, Ai and co workers fabricated an amperometric biosensor by binding AChE and quantum dot onto graphene-chitosan nanocomposite
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functionalized glassy carbon electrode.20 Gong and coworkers reported a flow injection/amperometric biosensor for pesticides assay on the basis of immobilization of AChE on layered double hydroxides.21 Although impressive progress has been made, the key problem in these methods is that the immobilization of enzymes usually leads to the impairment of the enzyme bioactivity. To overcome this problem, our group has been devoting to develop electrochemical biosensors that need no immobilization of AChE onto the electrode surface.22, 23 In our previous work, we have developed a new electrochemical biosensor based on the combination of AChE catalysis and signal amplification via hybridization chain reaction (HCR) to realize sensitive detection of pesticide residues.22 However, as AChEcatalyzed hydrolysis product-triggered DNA configurational change and HCR reaction occur on the solution-electrode interface, the spatial hindrance and the loss of configurational freedom usually make these heterogeneous analysis has relatively lower binding efficiency than that of the homogeneous analysis. Also, the need of immobilization of DNA onto the electrode surface is laborious and time-consuming, which restricts their practical application. Therefore, it is highly desirable to develop simple and sensitive immobilization-free homogeneous electroanalytical platform for AChE-based pesticide assay. Recently, a variety of homogeneous electroanalytical approaches have been designed to detect various targets, such as DNA, metal ions, biological molecules, and enzyme activities.24-34 For example, Hsing and coworks have developed solution-phase electroanalytical methods for the determination of DNA and Hg2+.24-26 Our group has developed homogeneous electroanalytical strategies for highly sensitive detection of adenosine triphosphate (ATP),27 the activity of human telomerase28 DNA methyltransferase,29, 30 and alkaline phosphatase.31 Compared with heterogeneous approaches, the homogeneous electroanalytical strategies possess the merits of rapid response and high recognition efficiency. However, these assays usually require signal moleculelabeled oligonucleotides, which are expensive and inevitably limit their wider applications. Therefore, immobilization-free electrochemical methods with labelfree strategies would be a desirable option. Furthermore, to realize the sensitive detection of pesticides, it is of significance to rationally design signal amplification strategies. In this aspect, DNA-based amplification techniques have attracted much attention owing to their diverse signal amplification formats allowed for the development of homogeneous binding assays. For example, polymerase chain reaction, strand-displacement amplification, HCR, rolling circle amplification (RCA), etc have been successfully used for highly sensitive target analyte assays.35, 36 Among these amplification methods, RCA, a powerful isothermal amplification tool, has been widely employed as a signal amplification technique for the determination of DNA, RNA, and proteins. In
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particular, RCA combined with the recognition of padlock probe exhibited various advantages in biosensing applications owing to their unique properties of producing long single-stranded DNA (ssDNA) with thousands of repeated sequences complementary to the circularized padlock probe.37, 38 With these in mind, herein, we developed a new labelfree and immobilization-free homogeneous electroanalytical platform for the detection of organophosphorous and carbamate pesticides, which relies on the combination of AChE-catalyzed hydrolysis product-mediated DNA configurational change and signal amplification by RCA reaction. In the absence of pesticides, the generated enzyme hydrolysis product can change the conformation of helper probe (HP) from hairpin structure to single-stranded structure. This singlestranded HP then hybridized with the padlock probe, initiating the specific ligation and RCA reaction. The RCA-mediated formation of G-quadruplex units captured a large number of methylene blue (MB) molecules, thus leading to the reduced diffusion of MB to the surface of indium tin oxide (ITO) electrode and producing a significantly decreased electrochemical response. When pesticides are present, AChE activity was inhibited and the subsequent reaction was retarded. As a result, numerous free MB molecules in solution could diffuse to the negatively charged ITO electrode and produce a high electrochemical signal. Therefore, a “signal-on” homogeneous electroanalytical platform for pesticides detection was developed on the basis of its inhibition effect on AChE activity. To verify the performance of this assay, omethoate, a representative organophosphorous pesticide, was chosen as a model target. Moreover, this method was also applied to assay omethoate contained in real samples of fruits and vegetables. Therefore, the strategy we proposed here holds great promise for pesticides detection in the food safety field. EXPERIMENTAL SECTION Reagents and Apparatus. The reagents and apparatus used in this work are described in detail in the supporting information. Procedures for Pesticide Assay. All DNA probes were directly used and diluted by 50 mM Tris–HCl buffer solution (pH = 7.4, containing 10 mM MgCl2) to give the stock solution. Firstly, Hg2+and HP were mixed at a molar ratio of 1: n (n is the number of T-T mismatch) to generate hairpin structured HP via the incorporation of Hg2+ to T-T mismatch. For sensing pesticide, various concentrations of omethoate were incubated with 1 mU/mL AChE solution (50 mM Tris-HCl, 10 mM MgCl2, pH = 7.4) at 37 °C for 30 min. Then, 30 nM acetylthiocholine chloride (ATCh) was added and incubated at 37 °C for 100 min. Next, 10 μL of the obtained solution was added into the ligation reaction solution, containing 10 nM HP, 30 nM padlock probe, 1 U/μL T4 DNA ligase, and 1 mM ATP. The obtained solution was
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Figure 1. Principle of this label-free homogeneous electroanalytical method for pesticide assay. incubated at 37 °C for 1 h to form a circular template.37-40 Subsequently, RCA reaction was conducted at 30 °C in a 60 μL reaction mixture containing 40 μL of ligation reaction products, 50 mM Tris−HCl buffer (pH=7.4), 10 mM (NH4)2SO4, 50 mM KCl, 10 mM MgCl2, 500 μM dNTP, 0.08 U/μL Phi29 DNA polymerase, and 0.2 mg/mL bovine serum albumin (BSA).37-40 Finally, 10 μL of MB solution was added into the above solution and incubated at 37 °C for 15 min before the electrochemical detection. Detection of Omethoate in Real Samples. Peach and carrot, purchased from a local market (Qingdao, China), were selected as the real sample matrix to investigate the practical applicability of this method. The samples were first chopped and the edible parts were crushed well. Then, 5 g of each sample was mixed separately with 25 mL methanol in a beaker, and the mixtures were vigorously stirred for 6 h. Then, the resulted samples were centrifuged at 13000 rpm for 15 min. Subsequently, the supernatant was filtrated and spiked with different concentrations of omthoate. For each sample, a control sample (without spiking of omethoate) was also prepared using the above-mentioned procedure. Gel Electrophoresis Analysis. The reaction products of the system in the presence and absence of omethoate were analyzed in 0.8% agarose gel, which was run in 1 × TAE buffer at 100 V for 1 h. Then, the gel was stained by 1 × SYBR Green I for 30 min, followed by imaging on a Gel Doc XR+ Imaging System (BIO-RAD, USA). RESULTS AND DISCUSSION Principle of the Assay. Figure 1 illustrates the principle of the homogeneous electroanalytical platform for pesticides detection. The sensing system mainly comprises of HP, padlock probe, T4 DNA ligase, phi29 DNA polymerase, ATCh, AChE, and MB. The unmodified HP, which contains many T-T mismatches, can incorporate Hg2+ and fold into a hairpin structure by T– Hg2+–T base pair formation. The padlock probe is
designed to compose the hybridization sequence to HP and a C-rich sequence that is complementary to the sequence of G-quadruplex. The positively charged MB molecules, which can interact with G-quadruplex, act as common electrochemical indicators in this work. Previous studies have demonstrated that free MB molecules can generate a strong diffusion current in homogeneous solution, whereas the G-quadruplex “locked” MB molecules producing a weak diffusion current on the ITO electrode.41, 42 In the absence of pesticide molecules, ATCh molecules are catalyzed by AChE enzyme to produce thiocholine (TCh). The thiol group (–SH) contained in TCh makes it reactive with sequestered Hg2+ because of the reported higher affinity between –SH and Hg2+ than that of T–T mismatch.22, 23, 43 As a consequence, Hg2+ ions are released from the hairpin structured HP, generating many unfolded HP with single-stranded structure. Since the 5’and 3’-end of the padlock probe are complementary to the HP, it can then be ligated and circularized by using the single-stranded HP as a template and T4 DNA ligase as a tool enzyme. Once single-stranded HP and the padlock probe hybridized into a complex, RCA reaction is occurred in the presence of phi29 DNA polymerase and produces a long ssDNA molecule by replicating the padlock probe hundreds to thousands of times under isothermal conditions.37, 38 The RCA reaction product then folds into a series of G-quadruplex structures with the assistance of K+. Subsequently, the G-quadruplex structures can capture a large amount of MB molecules, leading to a significantly reduced diffusion of MB. Moreover, as the surface of ITO electrode and DNA chain both have negative charge, the electrostatic repulsion between them further prevents the “locked” MB molecules from reaching the electrode surface. Thus, a weak electrochemical current is recorded because of the presence of fewer free MB molecules in solution. On the contrary, when pesticide omethoate is present, it can bond to the active center of AChE and inhibit its
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activity. Therefore, less TCh is produced, leading to the releasing less Hg2+ initially sequestered in the hairpin structured HP. As a result, most of the HP maintain their hairpin structures in the reaction solution. This hairpin structured HP cannot hybridize with the padlock probe and trigger the following RCA reaction. With the addition of MB, a small portion of MB intercalates into the stems of HP, while the majority of MB is free in the solution, which shows high diffusivity toward the ITO electrode, thus generating a relatively large electrochemical signal. Using this “signal-on” mode, label-free and immobilization-free homogeneous electrochemical detection of pesticides is readily realized. Feasibility Study. To investigate the feasibility of this method, differential pulse voltammogram (DPV) response of the sensing system under different conditions was recorded and displayed in Figure 2A. Obviously, a very weak DPV current was observed when AChE was not incubated with omethoate (curve a). In contrast, a strong electrochemical signal was observed with the addition of omethoate (curve d). Moreover, in the absence of AChE, very high electrochemical signal was also obtained (curve f), indicating that the activity of AChE plays an important role in the proposed strategy. Without AChE-catalyzed hydrolysis reaction, Hg2+ embedded in the stem of HP could not be released. Thus, HP maintained their hairpin structures, which could not initiate the following RCA reaction. Numerous MB molecules were still in the free state and generated a high diffusion current. However, the single-stranded HP, which did not incorporated with Hg2+, could effectively trigger the RCA reaction, generating a series of G-quadruplex structures. Then, numerous MB molecules were “locked” into G-quadruplex, resulting in a significantly decreased electrochemical signal (curve b). This weak electrochemical signal is similar to that in curve a, suggesting that most of the Hg2+ was released from the HP in the absence of omethoate (curve a) due to the generation of a large amount of hydrolysis product TCh. Additionally, in control experiments where no padlock probe (curve e), T4 DNA ligase (curve c), or phi29 DNA polymerase (curve g) were added, strong electrochemical signals were also observed due to the lack of RCA reaction. Furthermore, the reaction products obtained from the system in the presence (lane 1) and absence (lane 2) of omethoate were analyzed by conducting 0.8% agarose gel electrophoresis (Figure 2B). Compared with lane 1, lane 2 shows a new bright band near the sample loading slot, which could be attributed to the RCA products because they were too long to move forward in the agarose gel, and suggested the occurrence of RCA reaction in the absence of pesticide. These results confirmed the feasibility of this homogeneous electroanalytical platform for the determination of pesticides. As the activity of enzyme can be affected by certain substances, it is necessary to eliminate the possibility of hindrance brought by pesticides and Hg2+ to the activity
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of T4 DNA ligase and phi29 DNA polymerase in our strategy. To simplify the operation steps, single-stranded HP without incorporation of Hg2+ was used to investigate the influence of pesticides on the activity of enzymes, and HP S1 containing no T-T mismatch that cannot incorporate with Hg2+ was used to test the influence of Hg2+ on the activity of enzymes. As shown in Figure S-1, when omethoate with different concentrations were incubated with T4 DNA ligase and phi 29 DNA polymerase, the electrochemical signal change ∆ip, which is the difference between the DPV peak currents of MB with the same concentration in buffer and in the presence of RCA reaction products, remained almost constant with omethoate concentrations increased from 0 to 10000 μg/L. Similar results were also observed by using the Hg2+ incubated enzymes (Figure S-2). These results indicate that the activity of T4 DNA ligase and phi29 DNA polymerase is not affected by omethoate or Hg2+.
Figure 2. (A) Differential pulse voltammograms (DPV) of the sensing system under different conditions: (a) in the absence and (d) presence of omethoate, (b) in the presence of single-stranded HP instead of hairpin structured HP, (c-g) in the absence of (c) T4 DNA ligase, and (e) padlock probe, (f) AChE, and (g) phi29 DNA polymerase, respectively. (B) Agarose gel electrophoresis image of the products obtained from the system in the presence (lane 1) and absence (lane 2) of omethate. Optimization of Experimental Conditions. Since the sensing strategy is based on HP conformational change triggered by Hg2+-release, it is important to optimize the number of T-T mismatches in the sequences of HP in order to obtain the best signal-to-noise ratio. Four HP sequences with the number of T-T mismatches ranging from 2 to 5 in the stem were designed and their electrochemical response in the absence (ip(blank)) and presence of omethoate (ip(omethoate)) were examined, respectively. Figure 3A shows the DPV peak current change ∆ip (∆ip = ip(omethoate) - ip(blank)), indicating that HP sequence with 3 T-T mismatches in the stem gives the biggest current change. The HP with 2 T-T mismatches could result in a relatively low ip(omethoate) owing to the weak stability of the HP, which could then trigger the formation of some G-quadruplex. However, more T-T mismatches contained in HP could induce a relatively high ip(blank), because more TCh were needed to open the hairpin structured HP. Under the same experiment
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condition, the number of the opened HP is thus reduced and led to the formation of less G-quadruplex and big electrochemical signals. Therefore, HP sequence with 3 TT mismatches could obtain the best signal-to-background ratio and was used in the following experiments. In addition to the number of T-T mismatch, some experimental conditions that can influence the detection performance were also optimized and the final electrochemical responses were measured without the addition of pesticide. Since AChE-catalyzed hydrolysis product plays an important role in this approach, which is dependent on its concentration and reaction time. We optimized the concentration and the reaction time of AChE. Figure 3B reveals the effect of AChE concentration on the electrochemical response of this sensing platform. Evidently, the DPV signals decreased significantly as the concentration of AChE increased from 0 to 1.0 mU/mL, but barely changed as the concentration further increased to 1.50 mU/mL, indicating that the optimum concentration for AChE is 1.00 mU/mL. Figure 3C displays the effect of AChE reaction time on this sensing platform. Obviously, the DPV signals decreased with the extension of AChE-catalyzed hydrolysis time up to 100 min, while further extending the reaction time to 150 min led to little change in the electrochemical signal. Thus, 1.0 mU/mL and 100 min were selected as the optimal AChE concentration and catalyzed hydrolysis time, respectively. Moreover, the effect of ATCh concentration on the DPV response of this system was also investigated. As shown in Figure 3D, as the concentration of ATCh increased from 0 to 40 nM, the DPV peak currents decreased gradually until ATCh concentration reached 30 nM. Hence, the optimum ATCh concentration was chosen to be 30 nM.
Figure 3. (A) Comparison of the DPV peak current change (∆ip = ip(omethoate) - ip(blank)) of the system by using HP with different numbers of T-T mismatch. (B-D) DPV signals observed under (B) AChE with various concentrations: 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.5 mU/mL; (C) different AChE-catalyzed hydrolysis time: 0, 20, 40, 60, 80, 100, 120, and 150 min; (D) different ATCh concentrations: 0, 5, 10, 15, 20, 25, 30, 35, and 40 nM.
Other experimental conditions, such as the concentrations of T4 DNA ligase, phi29 DNA polymerase, and MB, as well as their reaction time were also optimized. The T4 DNA ligase concentration and the reaction time affect the RCA reaction. When T4 DNA ligase with different concentrations was used, the electrochemical signals were detected. As shown in Figure 4A, DPV signal decreased obviously as the concentration of T4 DNA ligase being increased from 0 to 1.0 U/μL. Afterward, slightly decreased peak currents were observed when the concentration of T4 DNA ligase exceeded 1.0 U/μL. Figure 4B presents the effect of enzyme reaction time on the DPV signal of this method. Evidently, the DPV signal decreased gradually as the reaction time of T4 DNA ligase increased from 0 to 60 min. However, the DPV peak currents were quite similar after further extending the reaction time. Therefore, the optimal T4 DNA ligase concentration and reaction time were 1.0 U/μL and 60 min, respectively. Phi29 DNA polymerase concentration and reaction time also influence the RCA reaction. To evaluate the influence of phi29 DNA polymerase concentration, 0, 20, 40, 60, 80, 100, and 120 mU/μL phi29 DNA polymerase were used, respectively. As shown in Figure 4C, with the increase of phi29 DNA polymerase concentration, DPV peak current decreased gradually until 80 mU/μL phi29 DNA polymerase was used. Figure 4D revealed that a lower DPV signal was observed when a longer reaction time was adopted. But the peak currents did not change much and levelled off after 120 min. Thus, the optimal concentration and reaction time for phi29 DNA polymerase were 80 mU/μL and 120 min, respectively.
Figure 4. DPV peak currents obtained under: (A) T4 DNA ligase with different concentrations: 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.5 U/μL; (B) different reaction time of T4 DNA ligase: 0, 10, 20, 30, 40, 50, 60, 70, and 80 min; (C) different phi29 DNA polymerase concentrations: 0, 20, 40, 60, 80, 100, and 120 mU/μL; (D) different reaction time of phi29 DNA polymerase: 0, 30, 60, 90, 120, 150, and 180 min.
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To ensure high sensitivity of this biosensing strategy, sufficient amounts of MB molecules are needed to generate high electrochemical signal. However, if the concentration of MB was too high, it would cause high background current. To get the best performance of the proposed assay, the MB concentration was investigated by analyzing the change of DPV peak current ∆ip, which is the difference between the DPV peak currents in the presence and absence of omethoate. As displayed in Figure S-3, ∆ip exhibited a sharp increase as the MB concentration increased from 1 μM to 5 μM, but, with the MB concentration further increased to 9 μM, ∆ip only showed a slight elevation. In addition, the electrochemical signal of the biosensor is negatively related to the amount of MB intercalated in the Gquadruplex, so the time taken for the intercalation of MB molecules was also investigated and plotted in Figure S-4. Obviously, the DPV signals decreased progressively as the intercalation time increased from 0 to 15 min. However, the DPV signals did not change much, when the intercalation time was further extended to 21 min. Thus, 5 μM and 15 min were employed as the optimal MB concentration and intercalation time. Sensitivity for Omethoate Detection. Under the optimal sensing conditions, omethoate was utilized as a model pesticide to investigate the applicability of the sensing platform for the quantitative electrochemical detection of pesticides. The analytical performance was evaluated by adding different concentrations of omthoate into the sensing system and the electrochemical response was presented in Figure 5A. As expected, the DPV current gradually increased by increasing the concentration of omethoate. This suggested the efficient inhibition of AChE activity by omethoate, which further induces less RCA reaction and more MB molecules in free state to generate high diffusion current. The relationship between omethoate concentration and the DPV peak current is presented in Figure 5B. Evidently, omethoate concentrations ranging from 10 to 10000 μg/L could be directly detected by this homogeneous electroanalytical strategy. Furthermore, the DPV peak current is linearly dependent on the logarithm of omethoate concentrations ranging from 10 to 10000 μg/L (inset of Figure 5B). The correlation equation was determined to be ip = 253.5 log Comethoate −153.3, with the correlation coefficient of R2 = 0.996. Here, ip represents the DPV peak current, Comethoate is omethoate concentration. The limit of detection (LOD) was estimated to be 2.1 μg/L (based on S/N = 3), which is much lower than the maximum level permitted by Chinese National Standards (20 μg/kg). Additionally, this sensitivity is superior or comparable to that of several previously reported methods (Table S-2). According to the mechanism of the sensing strategy, this approach is expected to be effective for other organophosphate and carbamate pesticides, which can inhibit AChE activity. To verify this, we tested the electrochemical signals of this sensing system with the
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addition of other conventional pesticides, namely aldicarb, dibrom, diazinon, dursban, glyphosate, methomyl, carbaryl, and the mixture of same concentrations of omethoate and carbaryl, respectively. As shown in Figure S-5, the blank sample without addition of pesticide gave a very small peak current, while significantly enhanced peak currents were observed from the samples after adding these pesticides or the mixture. These results indicate that this homogeneous electroanalytical platform is applicable for the determination of other pesticides that can inhibit AChE activity.
Figure 5. (A) DPV currents of the sensing system after adding different concentrations of omethoate (a-n): 0, 10, 20, 40, 60, 100, 200, 400, 600, 1000, 2000, 4000, 6000, and 10,000 μg/L. (B) DPV peak currents plotted against the concentrations of omethoate. Inset: The linear relationship between DPV peak currents and the logarithm of omethoate concentrations (from 10 to 10,000 μg/L). Determination of Pesticide in Real Samples. The practical applications of this approach for real sample assay was investigated by monitoring omethoate in peach and carrot samples, which were purchased from the local market. Because the content of omethoate in the samples was lower than the LOD of this method, the validation procedure was performed by spiking the samples with 10, 100, 500, and 1000 μg/L omethoate, respectively. Then, the spiked samples were analyzed and the results are summarized in Table S-3. The designed system exhibited good recovery ranging from 97.7% to 110.7% for omethoate spiked real samples, which is in the recovery range permitted by Chinese National Standards (GB/T 27404-2008). Thus, the proposed homogeneous
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electroanalytical platform exhibits great promise for practical applications. CONCLUSIONS In summary, a label-free and immobilization-free homogeneous electroanalytical platform was proposed for sensitive “sign-on” detection of pesticide, which combines AChE-catalyzed reaction-mediated DNA conformational change and signal amplification via RCA reaction. The calibration curve exhibited a good linear range from 10 to 10,000 μg/L and a LOD as low as 2.1 μg/L for omethoate was achieved. Furthermore, satisfactory results were obtained by using this strategy to detect pesticides contained in peach and carrot samples. Therefore, the proposed strategy holds great promise for pesticide detection and can be further exploited for sensing applications in environment and food safety field.
ASSOCIATED CONTENT Supporting Information. (1) Experimental Section: Reagents, Apparatus, Effect of Pesticide on Enzyme Activity, 2+ Effect of Hg on Enzyme Activity, Versatility Study; (2) Figure S-1 to Figure S-5; (3) Table S-2: Comparison of the present method and reported methods. (4) Table S-3: Real sample detection results. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author * E-mail:
[email protected] Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (Nos. 21405089, 21375072, and 21575074), Scientific Research Award Fund for Excellent Middle-Aged and Young Scientists of Shandong Province (No. BS2014CL004), the Open Funds of the State Key Laboratory of Electroanalytical Chemistry (SKLEAC201710), the Research Foundation for Distinguished Scholars of Qingdao Agricultural University (No. 663-1114304), and the Special Foundation for Taishan Scholar of Shandong Provence (No. ts201511052).
REFERENCES (1) Aragay, G.; Pino, F.; Merkoci, A. Nanomaterials for Sensing and Destroying Pesticides. Chem. Rev. 2012, 112, 5317–5338. (2) Miao, Y.; He, N.; Zhu, J. J. History and New Developments of Assays for Cholinesterase Activity and Inhibition. Chem. Rev. 2010, 110, 5216–5234. (3) Mileson, B. E.; Chambers, J. E.; Chen, W. L.; Dettbarn, W.; Ehrich, M.; Eldefrawi, A. T.; Gaylor, D. W.; Hamernik, K.; Hodgson, E.; Karczmar, A. G.; Padilla, S.; Pope, C. N.; Richardson, R. J.; Saunders, D. R.; Sheets, L. P.; Sultatos, L. G.; Wallace, K. B. Common Mechanism of Toxicity: A Case Study of Organophosphorus Pesticides. Toxicol. Sci. 1998, 41, 8–20.
(4) Barker, Z.; Venkatchalam, V.; Martin, A. N.; Farquar, G. R.; Frank, M. Detecting Trace Pesticides in Real Time Using Single Particle Aerosol Mass Spectrometry. Anal. Chim. Acta. 2010, 661, 188–194. (5) Lee, J.; Lee, H. K. Fully Automated Dynamic In-Syringe Liquid-Phase Microextraction and On-Column Derivatization of Carbamate Pesticides with Gas Chromatography/Mass Spectrometric Analysis. Anal. Chem. 2011, 83, 6856–6861. (6) Brito, M. N.; Navickiene, S.; Polese, L.; Jardim, E. F. G.; Abakerli, B. R.; Ribeiro, L. M. Determination of Pesticide Residues in Coconut Water by Liquid–Liquid Extraction and Gas Chromatography with Electron-Capture Plus Thermionic Specific Detection and Solid-Phase Extraction and HighPerformance Liquid Chromatography with Ultraviolet Detection. J. Chromatogr. A. 2002, 957, 201–209. (7) Pavlov, V.; Xiao, Y.; Willner, I. Inhibition of the Acetycholinesterase-Stimulated Growth of Au Nanoparticles: Nanotechnology-Based Sensing of Nerve Gases. Nano Lett. 2005, 5, 649–653. (8) Ton, X. A.; Bui, S. T. B.; Resmini, M.; Bonomi, P.; Dika, I.; Soppera, O.; Haupt, K. A Versatile Fiber-Optic Fluorescence Sensor Based on Molecularly Imprinted Microstructures Polymerized in-situ. Angew. Chem. Int. Ed. 2013, 52, 8317–8321. (9) Gill, R.; Bahshi, L.; Freeman, R.; Willner, I. Optical Detection of Glucose and Acetylcholinesterase Inhibitors by H2O2-Sensitive CdSe/ZnS Quantum Dots. Angew. Chem. Int. Ed. 2008, 47, 1676–1679. (10) Scenza, D. J.; Levine, M. Selective Detection of NonAromatic Pesticides via Cyclodextrin-Promoted Fluorescence Modulation. New J. Chem. 2016, 40, 789–793. (11) Qian, G.; Wang, L.; Wu, Y.; Zhang, Q.; Sun, Q.; Liu, Y.; Liu, F. Q. A Monoclonalantibody-Based Sensitive Enzyme-Linked Immunosorbent Assay (ELISA) for the Analysis of the Organophosphorous Pesticides Chlorpyrifos-methyl in Real Samples. Food Chem. 2009, 117, 364–370. (12) Yao, G. H.; Liang, R. P.; Huang, C. F.; Wang, Y.; Qiu, J. D. Surface Plasmon Resonance Sensor Based on Magnetic Molecularly Imprinted Polymers Amplification for Pesticide Recognition. Anal. Chem. 2013, 85, 11944–11951. (13) Du, X. J.; Jiang, D.; Hao, N.; Liu, Q.; Jing, Q.; Dai, L. M.; Mao, H. P.; Wang, K. An ON1–OFF–ON2 Electrochemiluminescence Response: Combining the Intermolecular Specific Binding with A Radical Scavenger. Chem. Commun. 2015, 51, 11236–11239. (14) Gong, J. M.; Wang, X. Q.; Li, X.; Wang, K. W. Highly Sensitive Visible Light Activated Photoelectrochemical Biosensing of Organophosphate Pesticide Using Biofunctional Crossed Bismuth Oxyiodide Flake Arrays. Biosen. Bioelectron. 2012, 38, 43–49. (15) Carmen C.; Mayorga-Martinez, P. F.; Kurbanoglu, S.; Rivas L.; Ozkanb, S. A.; Merkoçi, A. Iridium Oxide Nanoparticle Induced Dual Catalytic/Inhibition Based Detection of Phenol and Pesticide Compounds. J. Mater. Chem. B. 2014, 2, 2233–2239. (16) Drechsel, L.; Schulz, M.; Stetten, F.; Moldovan, C.; Zengerle, R.; Paust, N. Electrochemical Pesticide Detection with Auto Dip –A Portable Platform for Automation of Crude Sample Analyses. Lab Chip 2015, 15, 704–710. (17) Wang, Y.; Zhang, S.; Du, D.; Shao, Y. Y.; Li, Z. H.; Wang, J.; Engelhard, M. H.; Li, J. H.; Lin, Y. H. Self Assembly of Acetylcholinesterase on a Gold Nanoparticles–Graphene Nanosheet Hybrid for Organophosphate Pesticide Detection Using Polyelectrolyte as a Linker. J. Mater. Chem. 2011, 21, 5319−5325. (18) Liang, R. N.; Song, D. A.; Zhang, R. M.; Qin, W. Potentiometric Sensing of Neutral Species Based on a Uniform-
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Sized Molecularly Imprinted Polymer as a Receptor. Angew. Chem. Int. Ed. 2010, 49, 2556–2559. (19) Sun, X. L.; Zhang, L. J.; Zhang, H. X.; Qian, H.; Zhang, Y. Z.; Tang, L. L.; Li, Z. J. Development and Application of 3-Chloro1,2-propandiol Electrochemical Sensor Based on a Polyaminothiophenol Modified Molecularly Imprinted Film. J. Agric. Food Chem. 2014, 62, 4552−4557. (20) Dong, J.; Zhao, H.; Qiao, F. M.; Liu, P.; Wang, X. D.; Ai, S. Y. Quantum Dot Immobilized Acetylcholinesterase for the Determination of Organophosphate Pesticides Using GrapheneChitosan Nanocomposite Modified Electrode. Anal. Methods. 2013, 5, 2866–2872. (21) Gong, J. M.; Guan, Z. Q.; Song, D. D. Biosensor Based on Acetylcholinesterase Immobilized onto Layered Double Hydroxides for Flow Injection/Amperometric Detection of Organophosphate Pesticides. Biosens. Bioelectron. 2013, 39, 320– 323. (22) Yang, Y. M.; Liu, X. J.; Wu, M.; Wang, X. Z.; Hou, T.; Li, F. Electrochemical Biosensing Strategy for Highly Sensitive Pesticide Assay Based on Mercury Ion-Mediated DNA Conformational Switch Coupled with Signal Amplification by Hybridization Chain Reaction. Sens. Actuators B: Chem. 2016, 236, 597–604. (23) Wang, X. Z.; Dong, S. S.; Hou, T.; Liu, L.; Liu, X. J.; Li, F. Exonuclease I-Aided Homogeneous Electrochemical Strategy for Organophosphorus Pesticide Detection Based on Enzyme Inhibition Integrated with a DNA Conformational Switch. Analyst 2016, 141, 1830–1836. (24) Xuan, F.; Luo, X. T.; Hsing, I. M. Ultrasensitive SolutionPhase Electrochemical Molecular Beacon-Based DNA Detection with Signal Amplification by Exonuclease III-Assisted Target Recycling. Anal. Chem. 2012, 84, 5216−5220. (25) Xuan, F.; Luo, X. T.; Hsing, I. M. ConformationDependent Exonuclease III Activity Mediated by Metal Ions Reshuffling on Thymine-Rich DNA Duplexes for an Ultrasensitive Electrochemical Method for Hg2+ Detection. Anal. Chem. 2013, 85, 4586−4593. (26) Xuan, F.; Fan, T. W.; Hsing, I. M. Electrochemical Interrogation of Kinetically-Controlled Dendritic DNA/PNA Assembly for Immobilization-Free and Enzyme-Free Nucleic Acids Sensing. ACS Nano 2015, 9, 5027–5033. (27) Liu, S. F.; Wang, Y.; Zhang, C. X.; Lin, Y.; Li, F. Homogeneous Electrochemical Aptamer-Based ATP Assay with Signal Amplification by Exonuclease III Assisted Target Recycling. Chem. Commun. 2013, 49, 2335−2337. (28) Liu, X. J.; Li, W.; Hou, T.; Dong, S. S.; Yu, G. H.; Li, F. Homogeneous Electrochemical Strategy for Human Telomerase Activity Assay at Single-Cell Level Based on T7 ExonucleaseAided Target Recycling Amplification. Anal. Chem. 2015, 87, 4030−4036. (29) Li, W.; Liu, X. J.; Hou, T.; Li, H. Y.; Li, F. Ultrasensitive Homogeneous Electrochemical Strategy for DNA Methyltransferase Activity Assay based on Autonomous Exonuclease III-Assisted Isothermal Cycling Signal Amplification. Biosens. Bioelectron. 2015, 70, 304−309. (30) Wang, X. Z.; Liu, X. L.; Hou, T.; Li, W.; Li, F. Highly Sensitive Homogeneous Electrochemical Assay for Methyltransferase Activity Based on Methylation-Responsive Exonuclease III-Assisted Signal Amplification. Sens. Actuators B: Chem. 2015, 208, 575−580. (31) Zhang, L. F.; Hou, T.; Li, H. Y.; Li, F. A Highly Sensitive Homogeneous Electrochemical Assay for Alkaline Phosphatase Activity Based on Single Molecular Beacon-Initiated T7 Exonuclease Mediated Signal Amplification. Analyst 2015, 140, 4030−4036.
Page 8 of 9
(32) Liu, S. F.; Lin, Y.; Wang, L.; Liu, T.; Cheng, C. B.; Wei, W. J.; Tang, B. Exonuclease III-Aided Autocatalytic DNA Biosensing Platform for Immobilization-Free and Ultrasensitive Electrochemical Detection of Nucleic Acid and Protein. Anal. Chem. 2014, 86, 4008−4015. (33) Zhang, D. W.; Nie, J.; Zhang, F. T.; Xu, L.; Zhou, Y. L.; Zhang, X. X. Novel Homogeneous Label-Free Electrochemical Aptasensor Based on Functional DNA Hairpin for Target Detection. Anal. Chem. 2013, 85, 9378−9382. (34) Wei, X. F.; Ma, X. M.; Sun, J. J.; Lin, Z. Y.; Guo, L. H.; Qiu, B.; Chen, G. N. DNA Methylation Detection and Inhibitor Screening Based on the Discrimination of the Aggregation of Long and Short DNA on a Negatively Charged Indium Tin Oxide Microelectrode. Anal. Chem. 2014, 86, 3563−3567. (35) Zhang, H. Q.; Li, F.; Dever, B.; Li, X. F.; Le, X. C. DNAMediated Homogeneous Binding Assays for Nucleic Acids and Proteins. Chem. Rev. 2013, 113, 2812–2841. (36) Liu, H. Y.; Li, L.; Wang, Q.; Duan, L. L.; Tang, B. Graphene Fluorescence Switch-Based Cooperative Amplification: A Sensitive and Accurate Method to Detection MicroRNA. Anal. Chem. 2014, 86, 5487–5493. (37) Liu, H. Y.; Li, L.; Duan, L. L.; Wang, X.; Xie, Y. X.; Tong, L. L.; Wang, Q.; Tang, B. High Specific and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based Exponential Rolling Circle Amplification. Anal. Chem. 2013, 85, 7941−7947. (38) Deng, R. J.; Tang, L. H.; Tian, Q. Q.; Wang, Y.; Lin, L.; Li, J. H. Toehold-initiated Rolling Circle Amplification for Visualizing Individual MicroRNAs In Situ in Single Cells. Angew. Chem. Int. Ed. 2014, 53, 2389−2393. (39) Tang, L. H.; Liu, Y.; Ali, M. M.; Kang, D. K.; Zhao, W. A.; Li, J. H. Colorimetric and Ultrasensitive Bioassay Based on a Dual-Amplification System Using Aptamer and DNAzyme. Anal. Chem. 2012, 84, 4711−4717. (40) Wang, L. D.; Tram, K.; Ali, M. M.; Salena, B. J.; Li, J. H.; Li, Y. F. Arrest of Rolling Circle Amplification by Protein-Binding DNA Aptamers. Chem. J. Eur. 2014, 20, 2420-2424. (41) Zhang, F. T.; Nie, J.; Zhang, D. W.; Chen, J. T.; Zhou, Y. L.; Zhang, X. X. Methylene Blue as a G-Quadruplex Binding Probe for Label-Free Homogeneous Electrochemical Biosensing. Anal. Chem. 2014, 86, 9489−9495. (42) Hou, T.; Li, W.; Liu, X. J.; Li, F. Label-Free and EnzymeFree Homogeneous Electrochemical Biosensing Strategy Based on Hybridization Chain Reaction: A Facile, Sensitive, and Highly Specific MicroRNA Assay. Anal. Chem. 2015, 87, 11368–11374. (43) Wang, X. Z.; Hou, T.; Dong, S. S.; Liu, X. J.; Li, F. Fluorescence Biosensing Strategy Based on Mercury IonMediated DNA Conformational Switch and Nicking EnzymeAssisted Cycling Amplification for Highly Sensitive Detection of Carbamate Pesticide. Biosens. Bioelectron. 2016, 77, 644–649.
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