Label-free hairpin-like aptamer and EIS-based practical, biostable sensor for acetamiprid detection.

Acetamiprid (ACE) is a kind of broad-spectrum pesticide that has potential health risk to human beings. Aptamers (Ap-DNA (1)) have a great potential as analytical tools for pesticide detection. In this work, a label-free electrochemical sensing assay for ACE determination is presented by electrochemical impedance spectroscopy (EIS). And the specific binding model between ACE and Ap-DNA (1) was further investigated for the first time. Circular dichroism (CD) spectroscopy and EIS demonstrated that the single strand AP-DNA (1) first formed a loosely secondary structure in Tris-HClO4 (20 mM, pH = 7.4), and then transformed into a more stable hairpin-like structure when incubated in binding buffer (B-buffer). The formed stem-loop bulge provides the specific capturing sites for ACE, forming ACE/AP-DNA (1) complex, and induced the RCT (charge transfer resistance) increase between the solution-based redox probe [Fe(CN)6]3-/4- and the electrode surface. The change of ΔRCT (charge transfer resistance change, ΔRCT = RCT(after)-RCT(before)) is positively related to the ACE level. As a result, the AP-DNA (1) biosensor showed a high sensitivity with the ACE concentration range spanning from 5 nM to 200 mM and a detection limit of 1 nM. The impedimetric AP-DNA (1) sensor also showed good selectivity to ACE over other selected pesticides and exhbited excellent performance in environmental water and orange juice samples analysis, with spiked recoveries in the range of 85.8% to 93.4% in lake water and 83.7% to 89.4% in orange juice. With good performance characteristics of practicality, sensitivity and selectivity, the AP-DNA (1) sensor holds a promising application for the on-site ACE detection.


Introduction
In the past decades, pesticides have been widely used to control, kill or repel pests for improving the quality of agricultural products [1,2]. However, the wide use of pesticides has led to inevitable consequences for Agro-products and environmental pollution [3]. Therefore, the a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 selectivity and sensitivity depending on ΔR CT with a detection limit of 1 nM. In addition, the Ap-DNA (1) film was successfully performed in environmental lake water and orange juice samples with good recoveries.
10 μM DNA stock solution was first prepared in 20 mM Tris-HCl (pH = 7.4), and heated at 80˚C for 5 min before cooling down to room temperature. After completion of DNA annealing reaction, 1 μM DNA immobilization buffer (I-DNA) containing 1.0 M NaCl, 1mM EDTA, 1 mM TCEP in Tris-HCl (10 mM, pH 8.0) and the binding buffer (B-buffer) containing 100 mM NaCl, 200 mM KCl, 5 mM MgCl 2 , 1 mM EDTA in Tris-HCl (20 mM, pH = 7.4) were subsequently prepared [2,38]. Different concentrations of ACE solutions were prepard with the B-buffer. Tropicana orange juice samples were bought in the Chaoshifa supermarket lacated at Zhanghua South Road. Lake water was sampled in the Summer Palace lake (S1 Fig in S1 File) and then filtered with the 0.45 μm filter membrane prior to the ACE "B-buffer" preparation. All other solutions were prepared with Milli-Q water (18.2 MO cm resistivity) if not specified.

Fabrication of the DNA sensor
The electrochemically activated gold electrodes and the thiolated AP-DNA (1) modified electrode films were prepared according to previous methods reported before [40,42]. After that, the AP-DNA (1) modified sensors were washed with 20 mM Tris-HCl (pH = 7.4) buffer and kept at 4˚C prior to use. Each AP-DNA (1) modified electrode can be used only one time.

Electrochemical measurements
CHI 650D electrochemical workstation (Shanghai Chenhua instrument Co. Ltd., China) was employed to perform EIS measurements with three-electrode system in an electrochemical cell containing 2 mM [Fe(CN) 6 ] 3-/4-Tris-NaClO 4 (20 mM, pH = 7.4) solution. Briefly, the AP-DNA (1) modified films were incubated with the ACE for certain durations of time, then washed with 20 mM Tris-NaClO 4 (pH = 7.4) buffer for 30 s to remove any unbound analyte. EIS of the AP-DNA (1) films before and after interaction with ACE were all recorded. All measurements were performed in triplicate.

Circular circular dichroism spectra measurement
Circular dichroism (CD) spectra measurement was carried out with the Jasco J-810 spectropolarimeter. First, preparing 25 μM AP-DNA (1) solutions in Tris-HCl (20 mM, pH = 7.4) buffer and B-buffer, respectively, then adding 25 μM ACE to the above prepared AP-DNA (1) solutions. Finally, measuring the CD spectra of the prepared two systems after 2 h. The spectra of each sample were accumulated and averaged from three scans. Parameters: λ ranged from 200 to 400 nm, intervals 0.5 nm, scan rate 200 nm min -1 .

ACE sensing principle
A schematic diagram illustrating the ACE electrochemical sensing principle was shown in Scheme 1. As shown in Scheme 1, the arbitary single strand AP-DNA (1) anchored to the gold electrode surface can form a loosely secondary structure in Tris-HClO 4 (20 mM, pH = 7.4), which cannot specifically bind with ACE. However, when incubated in B-buffer solution the loosely secondary structure or the arbitary single strand AP-DNA (1) transformed into a more stable hairpin-like structure, forming a stem-loop bulge in the AP-DNA (1), which can serve as the specific recognizing site for ACE [11]. The specific binding interaction between the stem-loop bulge and ACE formed a ACE/AP-DNA (1) complex that hindered the electron transfer and induced the chargetransfer resistance (R CT ) increase between the solution-based redox probe [Fe(CN) 6 ] 3−/4− and the electrode surface. The change of ΔR CT (ΔR CT = R CT(after) -R CT(before) ) is positively related to the ACE level, so the determination of ACE can be quantitatively tested by EIS.

Demonstration of the sensing principle
To capture the target molecule, aptamers must change its conformation to a secondary structure with selectively binding site [43], and then binding with the target molecules by forming Scheme 1. Principle of structure switchable AP-DNA (1) assay for the detection of ACE.
https://doi.org/10.1371/journal.pone.0244297.g001 many weak bonds (such as hydrogen bond interaction and π-stacking interaction) [44]. Studies showed that the CD spectrum can provide a reliable determination of the DNA conformation, and has been widely applied as a very useful tool to study the conformations of DNA in solution. So, CD measurements were first carried out to investigate the proposed sensing mechanism by investigating the conformational switches of the Ap-DNA (1). As shown in Fig  1, the arbitary control DNA (2) showed a negative peak around 235 nm and a positive peak at 272 nm in Tris-HClO 4 (20 mM, pH = 7.4) (line a), which is the characteristic spectrum of a random coiled single strand DNA as reported before [45]. For AP-DNA (1), the CD spectra showed a positive peak at 277 nm and a negative peak at 250 nm in Tris-HClO 4 (20 mM, pH = 7.4) (line b), which is the characteristic spectrum of a hairpin-like DNA (containing stem-loop structure) [46]. When instead by B-buffer, a notable increase was observed in the CD intensity at the negative (250 nm) and positive (277 nm) band (line d), proving the random coiled AP-DNA (1) formed a more stable hairpin-like structure. These results indicated that the AP-DNA (1) undergoes a structural change after incubating in Tris-HClO 4 /B-buffer, repectively, forming specific binding site for ACE. However, only minimal CD intensitiy changes at negative 250 nm and positive 277 nm were observed in the ACE/AP-DNA (1) Tris-HClO 4 system (curve c), demonstrating that binding with ACE has almost no effect on the hairpin structure. Similarly, very small CD change was observed for the ACE/AP-DNA (1) Bbuffer system (curve e). Therefore, we can infer that B-buffer can stabilize the hairpin-like structure of the AP-DNA (1), offering the binding site for ACE, but the binding interaction between ACE and AP-DNA (1) can no further induce obviously structural change [47].
Next, the specific binding interaction between ACE and AP-DNA (1) was demonstrated by EIS measurements. First, The AP-DNA (1) modified electrode were prepared by immersing the electrodes into the 1 μM AP-DNA (1)   [Fe(CN) 6 ] 3-/4-Tris-NaClO 4 (20 mM, pH = 7.4) solution to track the ACE binding processes, and the representative Nyquist plots for the corresponding films were shown Fig 2A. The impedance spectra were analyzed by using the modified Randles' equivalent circuit (inset of Fig 2A).
As shown in Fig 2A, the R CT was significantly increased when the prepared Ap-DNA (1) films were incubated in B-buffer (containg 50 nM ACE) for 40 min, confirming the specific binding interaction between the AP-DNA (1) film and ACE. This can be explained that the Ap-DNA (1) with more stable hairpin-like structure on the electrode surface provided the specfic binding sites for ACE, and the formed ACE/AP-DNA (1) complexes then hindered the electron transfer from the solution to the electrode surface [23] (enhanced the repulsion of the redox probe [Fe(CN) 6 ] 3-/4-), resulting in an increased R CT [38,48]. As a control, EIS experiments were also carried out by exploring the DNA (2) modified films reacting with 500 nM ACE in B-buffer (the same procedure as the Ap-DNA (1) film). The representative Nyqiust plots were analyzed and shown in Fig 2B. As shown in Fig 2B, just as expected, there showed only a very minor increase of ΔR CT after the control DNA (2) films were utilized over the same time, which can be neglected in camparison with the ACE induced Ap-DNA (1) films ΔR CT . Therefore, we can infer that the obviously increased ΔR CT of the Ap-DNA (1) film was attributed to the specificly capturing ACE by the formed hairpin-like binding sites (stem-loop bulge) on the electrode surface.
Form the above results, we can conclude that: (1) the thiolated single strand Ap-DNA (1) was first modified on the electrochemically activated gold electrodes surface via Au-S bond; (2) the immobilized Ap-DNA (1) folds into a second structure (loosely hairpin-like structure) on the electrode surface in Tris-HClO 4 (20 mM, pH = 7.4); (3) the loosely hairpin-like structure of the Ap-DNA (1) switched to a more stable hairpin-like structure when incubated in Bbuffer; (4) the stem-loop bulge of the hairpin-like Ap-DNA (1), acted as the ACE binding sites, can capture ACE onto the electrode surface and form ACE/AP-DNA (1) complexes, as shown in Scheme 1. Due to the formation of ACE/AP-DNA (1) complexes, R CT of the AP-DNA (1) film was greatly increased. Hence, by using Ap-DNA (1) as the specific recognition probe and ΔR CT as the output signal, ACE can be electrochemically quantified.

EIS detection of ACE
Subsequently, the Ap-DNA (1) modified films were utilized for determination of ACE by EIS. The representative Nyquist plots of the specific binding interaction between the Ap-DNA (1) films with 50 nM ACE were measured and shown in Fig 2. The measured impedance spectra were analyzed with the modified Randles' equivalent circuit (inset of Fig 2). The fitting results were calculated and listed in Table 1.
R s (solution resistance) is the resistance between the reference electrode and the Ap-DNA (1) films [23], ranging from 5.5 (0.26) O�cm 2 to 6.1 (0.27) O�cm 2 . C film accounts for the capacitance of the Ap-DNA (1) films on the working electrodes [49], shown in Table 1. It showed that the C film decreased after immersing the Ap-DNA (1) films in ACE/B-buffer solution, revealing that the Ap-DNA (1) films binding with ACE might lead to an increase in the film thickness that resulted in a decreased dielectric constant [23]. R x and the CPE (constant phase element) accounts for the behavior of the 6-MCH on the working electrode surfaces [50]. CPE accounts for the inhomogeneity of the films on the working electrode surface with the exponential modifier n = 0.9 [51]. For ACE quantification, R CT is the most important parameter and it represents the charge transfer resistance between the redox probe [Fe(CN) 6 ] 3-/4and the gold working electrode surface [52,53]. As shown in Table 1, after incubating the Ap-DNA (1) films with 50 nM ACE in B-buffer for 40 min, the R CT sharply increased from 1069.2 (16) O�cm 2 (R CT(before) ) to 1413.8 (13) O�cm 2 (R CT(after) ) with a ΔR CT of 344.7 (26.2) O�cm 2 .
Next, ΔR CT was used as the parameter and applied for the ACE detection. First, we exploited the ΔR CT changes of the Ap-DNA (1) films after incubation in ACE/B-buffer with increasing the ACE concentrations from 1.0 nM to 600 nM. As shown in Fig 3A, there was a  dramatic increase in the ΔR CT with increasing the ACE concentrations from 1.0 nM to 200 mM, and then the ΔR CT increased slowly from 200 nM to 600 nM. The results indicated that the higher ACE concentration used, the more ACE/AP-DNA (1) complexes formed on the electrode. On the contrary, the ΔR CT decreased as decreasing the ACE concentration. No obvious change of ΔR CT was observed when decreased to 1.0 nM ACE (compared with the backgrand ΔR CT of buffer), and the low detection limit of 1.0 nM was determined. The linear relationship between ΔR CT and the concentrations of ACE was in the range of 5.0 nM to 100 nM (shown in Fig 3B), and the fitted regression equation was Y(ΔR CT ) = 64.4+5.53X (C ACE ) (R 2 = 0.9). Furthermore, the comparison between this method and other approaches for the detection of ACE is listed in Table 2. As shown in Table 2, the proposed assay has distinctive advantages of wide dynamic range, lower detection limit over the reported colorimetric, fluorescent approaches, and it avoids all the complicated modification steps, possessing the merits of simple preparation of the DNA film, less modification steps of the electrode over the nanomaterial modified electrochemical sensors, thus offers a simple, fast and low cost method for ACE detection. Most of all, the results demonstrated that this electrochemical Ap-DNA (1) sensor has great potential to be applied to the AEC detection.

Performance of selectivity and stability
Next, we further investigated the selectivity of the Ap-DNA (1) sensor taking ΔR CT of the Ap-DNA (1) films as the evaluating indicator. Four pesticides of methyl parathion, chlorpyrifos, dipterex and atrazine were selected, and EIS were carried out to measure R CT changes of the Ap-DNA (1) films before and after interacting with the four pesticides. As shown in Fig 4A, ACE caused a considerable increase in ΔR CT , while the ΔR CT changes were all very samll for the selected pesticides (methyl parathion, chlorpyrifos, dipterex and atrazine),demonstrating that the Ap-DNA (1) film showed good selectivity to ACE. As discussed above, the excellent selectivity of this sensor was attributed to the highly specific binding interaction of the stemloop bulge of Ap-DNA (1) with ACE. Furthermore, compared with the homogeneous assays such as colorimetric, chemiluminescent and fluorescent sensors, the inhomogeneous electrochemical sensor films could be rinsed before and after each step, and suffer less environmental interference [23], which can avoid the case that the signals of colorimetric, chemiluminescent and fluorescent may be quenched and masked by coexisting environment pollutants. The excellent ability of the impedimetric Ap-DNA (1) sensor to distinguish ACE from other pesticides added a novel dimension of selectivity to ACE. Au/MWCNT-rGONR modified electrode 5×10 −14~1 ×10 −5 0.017 fM [38] Aptamer/polyaniline and AuNPs modified electrode 2.5×10 −5~2 ×10 −5 86 nM [39] Aptamer/AuNPs modified electrode 5×10 −9~6 ×10 −7 1 nM [48] Aptamer modified electrode 5×10 −9~2 ×10 −7 1 nM This study Stability of the immobilized DNA on the electrode surface is one of the outstanding performances for a electrochemical sensor, so EIS of the Ap-DNA (1) films with different storage time (at 4˚C) were continually investigated. As shown in Fig 4B,

Testing in real samples
In order to validate the feasibility of the assay to detect ACE, the performance of the sensing platform was evaluated with environmental lake water and orange juice samples (purified with 0.45 μM membrane prior to analysis). The recoveries were evaluated by standard addition method, and the experiments were undertaken by spiking the real samples with known concentrations of ACE. As shown in Table 3, excellent spiked recoveries were achieved for ACE detection, with a range of 85.8% to 93.4% for lake water samples and 83.7% to 89.4% for orange  juice samples. These results implied that the constructed electrochemical Ap-DNA (1) films were stable, reliable and succesfully applied to real samples analysis, indicating potential application as a good alternative to environmental and food samples for the detection of ACE.
Furthermore, an investigation of the inter-day/intraday reliability of the AP-DNA (1) sensor was also performed to further confirm the analytical method. 50 and 100 nM concentrations of ACE in real samples were tested. As shown in Fig 5, there were no obvious differences between the calculated average values obtained from the two different methods (inter-day and intraday EIS measurements) for both the lake water and orange juice samples. These results confirmed the inter-day and intraday reliability of the sensor in real samples.

Conclusion
In this paper, a label-free impedimetric AP-DNA (1) sensor was reported for the detection of ACE with sensitivity and selectivity. The modified AP-DNA (1) on the electrode surface formed a loosely secondary structure in Tris-HClO 4 , and then switched to a more stable hairpin-like structure with stem-loop bulge in B-buffer, which acted as the ACE binding sites. As a result, with Ap-DNA (1) as a sensing probe, ACE was sensitively detected with a limit of 1 nM. Additionally, the Ap-DNA (1) sensor showed excellent ability to distinguish ACE from other pesticides and practicability to detect ACE in lake water and orange juice, with good recoveries in the range from 83.7% to 93.4%. This sensor is easy, reliable and convenient for the detection of ACE, and enables detection of ACE without any chemical modification or fluorescent labeling of the Ap-DNA (1), demonstrating the AP-DNA (1) sensor possessed great potential applications and promising platform for monitoring ACE in environmental and food samples.