Ultrasensitive electrochemical DNAzyme sensor for lead ion based on cleavage-induced template-independent polymerization and alkaline phosphatase amplification

Biosensors and Bioelectronics 83 (2016) 33–38

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Ultrasensitive electrochemical DNAzyme sensor for lead ion based on cleavage-induced template-independent polymerization and alkaline phosphatase amplification Shufeng Liu n, Wenji Wei, Xinya Sun, Li Wang Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, No.53, Rd. Zhengzhou, Qingdao, Shandong 266042, China

art ic l e i nf o

a b s t r a c t

Article history: Received 2 February 2016 Received in revised form 28 March 2016 Accepted 11 April 2016 Available online 11 April 2016

In this article, a simple, highly sensitive and selective electrochemical DNAzyme sensor for Pb2 þ was developed on the basis of a 8–17 DNAzyme cleavage-induced template-independent polymerization and alkaline phosphatase amplification strategy. The hairpin-like substrate strand (HP DNA) of 8–17 DNAzyme was firstly immobilized onto the electrode. In the presence of Pb2 þ and the catalytic strand of 8–17 DNAzyme, the HP DNA could be cleaved to expose the free 3′-OH terminal, which could be then utilized for the cascade operation by terminal deoxynucleotidyl transferase (TdTase) for the base extension to incorporate biotinylated dUTP (dUTP-biotin). The further conjugated streptavidin-labeled alkaline phosphatase (SA-ALP) then catalyzed conversion of electrochemically inactive 1-naphthyl phosphate (1NP) for the generation of electrochemical response signal. The currently fabricated Pb2 þ sensor effectively combines triply cascade amplification effects including cyclic Pb2 þ -dependent DNAzyme cleavage, TdTase-mediated base extension and enzymatic catalysis of ALP. An impressive detection limit of 0.043 nM toward Pb2 þ with an excellent selectivity could be ultimately obtained, which was superior than most of the electrochemical methods. Thus, the developed amplification strategy opens a promising avenue for the detection of metal ions and may extend for the detection of other nucleic acid-related analytes. & 2016 Elsevier B.V. All rights reserved.

Keywords: Electrochemical sensor Pb2 þ detection DNAzyme Terminal deoxynucleotidyl transferase

1. Introduction Lead ion (Pb2 þ ), as a severe environmental pollutant, has been commonly recognized to exert adverse effects on human health, especially on children. It has been revealed that a very small amount of lead ion could incur serious damage to the brain, kidneys, and nervous system (Lu et al., 2013; Needleman, 2004; Godwin, 2001; Schneider and Clark, 2013; Dalavoy et al., 2008). Accordingly, the development of sensors for the detection of Pb2 þ in environmental or biological samples is of considerable significance. The routine analytical techniques for Pb2 þ include atomic absorption spectrometry, mass spectrometry and atomic emission spectrometry, etc (Yang and Saavedra, 1995; Liu et al., 2000; Elfering et al., 1998; Rebocho et al., 2006). They could usually achieve the sensitive and accurate assay for Pb2 þ , but most of them involve the use of relatively cumbersome instruments or complicated operation procedures and thus are so inappropriate for on-site and real-time detection. To address this issue, some n

Corresponding author. E-mail address: [email protected] (S. Liu).

http://dx.doi.org/10.1016/j.bios.2016.04.026 0956-5663/& 2016 Elsevier B.V. All rights reserved.

promising techniques have been developed to monitor the levels of Pb2 þ such as fluorescence (Marbella et al., 2009; Shi et al., 2014; Huang and Liu, 2014), colorimetry (Kuo et al., 2015; Li et al., 2010, 2013), electrochemistry (Lin et al., 2011; Xu et al., 2014; Zhang et al., 2011), electrochemiluminescence (Ma et al., 2011; Zhu et al., 2009), etc. Although great advances have been made for the Pb2 þ detection based on these techniques, sometimes they are also confronted with the relatively complex design or synthesis procedures, or even unsatisfactory detection performance. Therefore, the development of new determination method or strategy for the sensitive and selective Pb2 þ detection is still a challenge. DNAzymes are a variety of catalytic DNA sequences selected by in vitro selection that possess high catalytic activities toward specific substrates (Breaker and Joyce, 1994). Also, they are more stable than protein and can work in the relatively harsh conditions such as high temperature and extreme pH. Owing to the specific requirement of metal cofactors for RNA-cleaving DNAzyme during in vitro selection process, it has been emerged for the sensor fabrication toward different metal ions for example Mg2 þ , Na þ , Pb2 þ , UO22 þ , and lanthanides (Santoro and Joyce, 1997; Torabi et al., 2015; Lee et al., 2008; Huang et al., 2014; Liu and Lu, 2004; Lan et al., 2010). The DNAzymes have often been utilized as the

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effective signal amplification means for enzyme-free and sensitive detection toward various targets such as DNA, microRNA, enzyme activity, small molecules, and so on (Zhao et al., 2013; Liu and Lu, 2007; Tian et al., 2012; Zhang et al., 2010; Kim et al., 2007). As one of the RNA-cleaving DNAzymes, 8–17 DNAzyme is a Pb2 þ -dependent DNAzyme and has been implemented to develop colorimetric, electrochemical and fluorescence sensors for Pb2 þ detection (Li et al., 2009; Wang et al., 2008; Xu et al., 2013; Tang et al., 2013). It should be noted that the DNAzyme-based detection strategies for Pb2 þ are more preferentially combined with the optical methods. By comparison, electrochemical method could offer significant advantages such as its inherent signal stability, low cost, high sensitivity and ease of calibration. Currently, the electrochemical DNAzyme-based Pb2 þ ion sensors could be simply categorized into two types. One is based on the conformation change of the redox-active group modified DNAzyme strand after the Pb2 þ -dependent cleavage, which is usually limited with the insufficient detection sensitivity (Xiao et al., 2007). Another one could be considered as post-amplification strategy after the Pb2 þ dependent specific cleavage process, which is more utilized toward the sensitive detection of Pb2 þ (Shen et al., 2008). For example, Yang et al. developed a novel amplification strategy for Pb2 þ detection based on DNAzyme functionalized gold nanoparticles (Yang et al., 2010). Cui et al. developed an electrochemical sensor for the amplified detection of Pb2 þ by a DNA functionalized porphyrinic metal-organic framework (Cui et al., 2015). Usually, the post-amplification strategies involve the relatively complex preparation or functionalization steps and sometimes the stability issue of the nanomaterial. Therefore, the development of new and easy to use signal amplification strategy for the sensitive and selective Pb2 þ determination is still highly desirable. Terminal deoxynucleotidyl transferase (TdTase), a templateindependent DNA polymerase that can catalyze the sequential addition of deoxynucleotides (dNTPs) at the 3′-OH group of nucleic acid strands (Rosemeyer et al., 1995; Lu et al., 2015). Unlike DNA polymerases, it does not require a template and has been widely employed for the extension of DNA in the solution or the fabrication of DNA nanostructure from a surface (Chow et al., 2005; Chow and Chilkoti, 2007; Khan et al., 2013). It can also be used to execute the labeling of the 3′-end of DNA probes with different tags for a variety of detection and affinity application (Tjong, et al., 2013; Wu et al., 2012; Chi et al., 2015; Anne et al., 2007; Yang et al., 2014). Considered with the operation flexibility and signal amplification capability of the TdTase, it was conceived to offer the promising opportunities for the amplified detection of analytes if the new 3′-OH terminal of nucleic acid could be created after target response. Herein, an electrochemical DNAzyme sensor for Pb2 þ detection was proposed, which was based on the Pb2 þ -dependent 8–17 DNAzyme cleavage-induced base extension by TdTase for signal amplification. The 8–17 DNAzyme was used for the selective recognition unit for Pb2 þ . The hairpin-like substrate DNA strand of 8–17 DNAzyme was firstly immobilized on the electrode and heretofore no exposed 3′-OH could be utilized as the primer of TdTase. In the presence of Pb2 þ and the catalytic strand of 8–17 DNAzyme, the HP DNA could be cleaved and the 3′-OH terminal could be generated and then operated by TdTase for the base extension to incorporate biotinylated dUTP (dUTP-biotin). The streptavidin-labeled alkaline phosphatase (SA-ALP) could be further conjugated with the extended biotin. The introduced ALP then catalyzes conversion of electrochemically inactive 1-naphthyl phosphate (1-NP) into an electrochemically active phenol for the generation of an amplified electrochemical signal (Carpini et al., 2004). Taking advantage of the TdTase-mediated base extension principle, the amplified detection of Pb2 þ could be achieved. Owing to no involvement of extra DNA sequence and also no

hybridization recognition-based amplification process during TdTase-mediated base extension process, the advised DNAzyme cleavage-induced template-independent polymerization strategy is thus relatively simple and direct for Pb2 þ determination. Also, the triply sequential amplification process would be beneficial for the Pb2 þ detection with a high confidence and sensitivity.

2. Experimental section 2.1. Materials and chemicals The hairpin-like 8–17 DNAzyme substrate strand (HP) 5′-SH(CH2)6-TTT TGC GCC GCC GCA AAA TTC ACC AAC TAT rA GGA AGA GAT GTT ACG AGG CGG CGG CGC TTT T-(CH2)6-SH-3′, the catalytic DNA strand of 8–17 DNAzyme 5′-CAT CTC TTC TCC GAG CCG GTC GAA ATA GTT GGT-PO4-3′ were synthesized by Sangon Biotech. Co., Ltd. (Shanghai, China). The terminal deoxynucleotidyl transferase (TdTase), biotin-11-dUTP and dUTP were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The 6-mercaptohexanol and 1-Naphthyl phosphate (1-NP) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Bovine serum albumin (BSA) and streptavidin-alkaline phosphatase (SA-ALP) were supplied by Dingguo Biotech Co., Ltd. (Beijing, China). The 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt (HEPES) was obtained from Sangon Biotech. Co., Ltd. (Shanghai, China). All of the other chemicals were of analytical reagent grade and used without further purification. 2.2. Apparatus The Differential pulse voltammetry (DPV) and electrochemical impedance spectra (EIS) experiments were performed on a CHI 660D electrochemical workstation (Shanghai, China). A conventional three-electrode system included a gold electrode as the working electrode, a platinum wire as the auxiliary electrode, and a Ag/AgCl reference electrode. 2.3. Immobilization of HP DNA on the electrode The gold electrode (2 mm diameter) was sequentially polished with 1.0 mm, 0.3 mm and 0.05 mm alumina power, followed by ultrasonic cleaning in acetone and water. Subsequently, the gold electrode was dipped in a freshly prepared piranha solution (a 3:1 v/v mixture of concentrated H2SO4 and 30% H2O2) for 10 min, and then rinsed thoroughly with water. Then, the electrochemical pretreatments were performed by cycling the potential between  0.2 and þ1.5 V in 0.5 M H2SO4 solution until the stable cyclic voltammogram was obtained. Finally, the electrode was washed with water and dried under a nitrogen stream. Each DNA solution was heated to 95 °C for 30 s and then allowed to cool to room temperature before use. The immobilization of HP DNA on the electrode was performed by incubation into 10 mM Tris–HCl (10 mM TCEP, 0.5 M NaCl, pH 7.4) containing 1 mM HP DNA for 12 h at room temperature. After incubation step, the electrode was rinsed with Tris–HCl buffer. Then, the modified electrode was immersed into 1 mM 6-mercaptohexanol (MCH) solution for 1 h to remove the nonspecific DNA adsorption. Then, the electrode was rinsed with Tris–HCl buffer. 2.4. Pb2 þ -dependent DNAzyme cleavage and TdTase-mediated extension The Pb2 þ -dependent DNAzyme cleavage reaction was operated by immersing the HP DNA assembled electrode into the 70 μL 25 mM HEPES buffer (200 mM NaCl, pH 7.0) containing 0.5 μM

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catalytic DNA strand of 8–17 DNAzyme and different concentration of Pb2 þ at 37 °C for 40 min. After rinsing with Tris–HCl buffer, the electrode was immersed in an extension solution (20 mM Tris– HCl, 50 mM KCl, 10 mM Mg(Ac)2, 0.25 mM CoCl2, pH 7.9, 200 U/mL TdTase, 10 μM dUTP-biotin) for 1.5 h at 37 °C. Then the electrode was immersed into a 2% BSA solution and incubated at 37 °C for 1 h to block the nonspecific site. Then, 20 μL 5 μg/mL SA-ALP solution was added onto the electrode surface and incubated at 37 °C for 40 min. 2.5. Electrochemical detection The Differential pulse voltammetric (DPV) experiments were recorded in 100 mM Tris–HCl buffer (1 mM Mg2 þ , pH 9.8) containing 4 mM 1-NP with the potential window from 0 V to 0.6 V, the pulse amplitude of 50 mV and the pulse period of 0.2 S. The electrochemical impedance spectra (EIS) were recorded in 5 mM [Fe(CN)6]3  /4  and 1 M KCl with the frequency range from 0.1 to 10 kHz. Before measurements, the electrolyte solution should be purged with nitrogen gas for about 20 min. The Pb2 þ detection in the tap water sample was operated according to the following procedures. The tap water was directly collected with no further processing, and then diluted with 25 mM HEPES buffer (200 mM NaCl, pH 7.0) to obtain the 5% tap water samples. The Pb2 þ ions with different concentrations (5, 10 and 100 nM) were added into the diluted tap water samples for the execution of the recovery experiments.

3. Results and discussion 3.1. Design of strategy 2þ

The fabricated electrochemical DNAzyme sensor for Pb detection was schematically illustrated in Fig. 1. It took advantage of the 8–17 DNAzyme as a selective recognition element and the terminal deoxynucleotidyl transferase (TdTase)-mediated base extension for signal amplification. The lead ion-specific 8–17 DNAzyme employed in this work was consisted of a catalytic DNA strand with a modified 3′-phosphoryl end and a hairpin-like (HP) DNA substrate strand with a single, sessile ribonucleoside adenosine (rA) (represented in red color in Fig. 1). The HP substrate strand was modified with mercapto groups at both ends and it could be firmly immobilized on the electrode surface by the Au–S

Fig. 1. Schematic illustration of electrochemical DNAzyme sensor for lead ion based on the TdTase-mediated extension and alkaline phosphatase amplification. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

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interaction. The catalytic DNA strand could be hybridized with the HP DNA substrate strand to form a stable duplex with the internal loop for the accommodation of Pb2 þ . In the presence of Pb2 þ , the 8–17 DNAzyme was activated and the HP substrate strand could be cleaved to expose the 3′-OH, which could be catalyzed by TdTase for the sequential addition of biotin-labeled dUTP onto one of the cleaved substrate strand (Rosemeyer et al., 1995; Lu et al., 2015). Simultaneously, the Pb2 þ could be released to participate for the successive cleavage process. After BSA blocking, the streptavidinlabeled alkaline phosphatase (SA-ALP) could be linked onto the electrode by its affinity interaction with biotin. The introduced ALP then catalyzed conversion of electrochemically inactive 1-naphthyl phosphate (1-NP) into an electrochemically active phenol for the generation of a remarkable electrochemical response. In the absence of Pb2 þ , the HP DNA substrate strand could not be cleaved. Also, the catalytic strand of 8–17 DNAzyme was functionalized with a phosphate group at 3′ terminal. Thus, no exposed 3′-OH could be utilized for the TdTase-mediated sequence extension and accordingly the SA-ALP would not be specifically linked onto the electrode for the electrochemical response. The currently proposed electrochemical sensor for lead ion was involved into three amplification elements toward Pb2 þ detection. Firstly, the Pb2 þ could be circularly participated into the 8–17 DNAzyme cleavage process. Secondly, the TdTase-mediated sequence extension was used for the introduction of a plentiful of biotin moiety onto the electrode surface for the further conjugation of SA-ALP. Thirdly, the enzymatic catalysis of ALP contributed for the further increased electrochemical response. Therefore, the current DNAzyme cleavage-induced template-independent polymerization and alkaline phosphatase amplification strategy was hopeful to offer an ultrahigh sensitivity for the assay of lead ion. 3.2. Feasibility of the fabricated electrochemical sensor for Pb2 þ detection The feasibility of the proposed DNAzyme cleavage-induced template-independent polymerization and alkaline phosphatase amplification strategy for Pb2 þ was firstly verified and shown in Fig. 2. In the presence of Pb2 þ , a distinct electrochemical oxidation signal at the potential of about 0.28 V was obtained (curve b in Fig. 2). However, only a relatively weak electrochemical response could be obtained in the case of no Pb2 þ (curve a in Fig. 2), which might be attributed to the non-specific adsorption of trace amounts of SA-ALP onto the electrode surface. The decreased

Fig. 2. Differential pulse voltammograms obtained at different experimental conditions. (a) no Pb2 þ , (b) 1 μM Pb2 þ , (c) no 8–17 DNAzymee, (d) no TdTase.

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about 4453 Ω was observed (curve c in Fig. 3), which might be ascribed to the easier accessibility of Fe(CN)63  /4  toward electrode surface owing to decreased repellence by the opened HP compared with the more densely packed structure of the caged HP on the electrode surface (Yang et al., 2012; Liu et al., 2013). Furthermore, after TdTase-mediated dUTP-biotin extension, the diameter of the semicircle increased evidently with the Ret value of about 6901 Ω (curve d in Fig. 3). After BSA blocking for the occupation of the nonspecific adsorption site, the Ret value was found to be further increased (curve e in Fig. 3). Furthermore, the affinity reaction for the binding of SA-ALP on the electrode surface evidently increased the electron transfer resistance with the Ret value of about 9131 Ω (curve f in Fig. 3). This could be easily understood that the bulky protein molecule binding on the electrode surface largely inhibited the approaching of Fe(CN)63  /4  toward electrode surface. The EIS experiments could further provide beneficial supports for Pb2 þ detection by the DNAzyme cleavageinduced template-independent polymerization and alkaline phosphatase amplification strategy. Fig. 3. Electrochemical impedance spectra (EIS) related with the differently modified electrodes. (a) bare gold electrode, (b) DNA probe/MCH modified electrode, (c) 8–17 DNAzyme and Pb2 þ cleavage, (d) TdTase-mediated dUTP-biotin extension, (e) BSA blocking for the nonspecific site, (f) the conjugation of the SA-ALP with the extended biotin. The inset shows the equivalent Randles electrical circuit.

electrochemical response compared with the background signal could be only obtained for the control experiment in the absence of catalytic strand of 8–17 DNAzyme (curve c in Fig. 2). Also, for the case of no TdTase, the electrochemical response was found to be further decreased (curve d in Fig. 2). We also used the dUTP as the substitute of biotin-dUTP during the TdTase-mediated extension process. The electrochemical response was almost same with the background value whether in the presence of Pb2 þ or not (Fig. S1), further indicating that electrochemical response was indeed arouse from the linked SA-ALP by affinity interaction. The electrode modification and sensor fabrication process was also confirmed by electrochemical impedance spectroscopy (EIS) measurements (Fig. 3). The EIS data were fitted by an equivalent electrical circuit (inset, Fig. 3). In EIS, the semicircle diameter could reflect the electron-transfer resistance, Ret, which dominates the electron transfer kinetics of the redox probe at the electrode interface. The bare gold electrode witnessed a very small Ret value of about 364 Ω (curve a in Fig. 3). After the assembly of the HP and MCH on the electrode, a big semicircle with Ret value of about 5067 Ω could be observed (curve b in Fig. 3), indicating an evident increase of the charge transfer resistance, which could be ascribed to the repellence of Fe(CN)63  /4  from approaching electrode surface by negative-charged phosphate skeletons of HP DNA. After Pb2 þ -dependent DNAzyme cleavage, a decreased Ret value of

3.3. Optimization of assay conditions In order to achieve the best sensing performance, the corresponding experimental conditions were optimized based on the DPV peak current change (ΔI) (ΔI ¼I  I0; I and I0 represented the currents in the presence and absence of Pb2 þ , respectively). It could be seen from Fig. 4A that the HP DNA concentration of 1 μM could achieve the best electrochemical response toward Pb2 þ detection. With the further increase of HP DNA concentration, the electrochemical responses toward Pb2 þ were observed with a gradual decrease. This might be attributed that the higher HP DNA assembly density on the electrode surface would be not beneficial for the Pb2 þ recognition or the electron transfer of the catalytically generated naphthol toward electrode surface. Thus the optimized concentration for HP DNA was chose as 1 μM. The DNAzyme cleavage time was further optimized. It could be seen from Fig. 4B that the ΔI increased with the cleavage time and almost reached the plateau at the cleavage time of 40 min. The optimized 8–17 DNAzyme cleavage time was determined as 40 min. The effects of the DNAzyme reaction temperature and pH on the electrochemical response toward Pb2 þ were also investigated (Fig. S2). The ΔI increased with the temperature and reached the maximum at about 37 °C (Fig. S2A), indicating the enhanced DNAzyme cleavage rate at the elevated temperature (Li and Lu, 2000). The distinctly decreased ΔI at a further elevated temperature might be explained that a higher temperature would not be beneficial for the formation of the enzyme-substrate complex. As shown from Fig. S2B, a higher ΔI could be obtained at pH 7.0 than that at pH 6.0,

Fig. 4. (A) Optimization of HP DNA immobilization concentration. The used HP DNA concentrations were 0.2, 0.5, 1.0, 2.0 and 5.0 μM, respectively; (B) Optimization of Pb2 þ dependent DNAzyme cleavage time; (B) Optimization of TdTase reaction time. The used Pb2 þ concentrations were all 1 μM. The error bars were obtained based on three repetitive experiments.

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Fig. 5. (A) Differential pulse voltammograms corresponding to the analysis of different concentrations of Pb2 þ . The concentrations of Pb2 þ for the curves from (a) to (j) are: 0, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500 nM. (B) The linear plot between the ΔI and the logarithm value of the Pb2 þ concentration from 0.05 to 500 nM. The error bars were obtained based on three repetitive experiments.

indicating an increased DNAzyme cleavage activity (Brown et al., 2003). The further increased pH value of 8.0 resulted in a slightly decreased electrochemical response toward Pb2 þ . It might be partially due to the high tendency for Pb2 þ to form precipitates at high pH value, reducing Pb2 þ concentration in the solution. The TdTase reaction time was also optimized and listed in Fig. 4C. The electrochemical response increased with the TdTase reaction time and the tendency became sluggish after 1.5 h. Herein the 1.5 h was chose for the TdTase-mediated extension time. Furthermore, the effect of TdTase concentration on the electrochemical response was investigated (Fig. S3). An adequate amount of 200 U mL  1 of TdTase was ultimately employed for the sufficient execution of TdTase-mediated based extension operation. 3.4. Detection sensitivity for the Pb2 þ Under the optimized experimental conditions, the detection performance of the fabricated electrochemical sensor was investigated by using Pb2 þ with different concentrations. As shown in Fig. 5A, the electrochemical intensity was observed to increase with the concentration of Pb2 þ , indicating that the DNAzyme cleavage and the accompanying TdTase-mediated extension were highly dependent on the concentration of Pb2 þ . A good linear relationship between the ΔI and the logarithm value of the Pb2 þ concentration ranged from 0.05 to 500 nM could be obtained (Fig. 5B). The regression equation was Y ¼  0.63  0.38 lgX (Y and X represented the ΔI and Pb2 þ concentration, respectively; unit of X was nM) with the correlation coefficient of 0.9980. The detection limit, which was defined as 3 times the standard deviation of the background, was about 0.043 nM. This detection limit is superior or comparable with that of the mostly reported Pb2 þ sensors (Tab. S1). The cutoff value of Pb2 þ ions in the drinkable water defined by the US Environmental Protection Agency is 72 nM (Yang et al., 2010) (http://www.epa.gov/safewater/contaminants/index.html, 1/30/2010). Thus, the developed sensors could completely meet the requirement of water-quality monitoring. 3.5. Selectivity, reproducibility and stability of the developed sensor The selectivity of the developed sensor for Pb2 þ was further investigated by using several metal ions including Mg2 þ , Cd2 þ , Cu2 þ , Mn2 þ , Hg2 þ , Fe3 þ , Ni2 þ , Zn2 þ and Ag þ (Fig. 6). It could be seen that all the investigated possible interfering ions only showed the negligible electrochemical responses (ΔI). Moreover, we challenged the Pb2 þ detection in the coexistence of other metal ions. As shown in Fig. S4, the coexistence of other tested metal ions

Fig. 6. Selectivity of the fabricated sensor for Pb2 þ compared to other interfering ions. The concentrations for Pb2 þ and other interfering metal ions were all 100 nM. The error bars were obtained based on three repetitive experiments.

(Mg2 þ , Mn2 þ , Hg2 þ , Zn2 þ ) had almost no obvious effect on the response of this sensor to Pb2 þ . These results indicated that the proposed sensing system was highly selective for Pb2 þ detection. The fabricated sensor showed the relative standard deviations of 5.3% and 5.8% for the detection of 100 nM and 1 μM Pb2 þ , respectively, based on five repetitive measurements, revealing a relatively good reproducibility of the proposed protocol. The stability for the HP DNA modified electrode has also been checked. Three independent experiments demonstrated that the HP DNA modified electrode could retain about 91.3% of its initial response toward 100 nM Pb2 þ after its storage in the refrigerator at 4 °C for 10 days, indicating a relatively robust stability of the modified electrode. In order to probe the applicative feasibility of the proposed sensing system in environment samples, the recovery experiments were carried out in the 5% diluted tap water samples. None of the existing Pb2 þ ions could be detected in the 5% diluted tap water sample by the fabricated electrochemical sensor. Then, Pb2 þ ions were added into the diluted tap water samples at three different concentrations of 5 nM, 10 nM and 100 nM. The analytical results were shown in Table S2. Recovery values ranging from 97% to 104% indicated that the established sensing system might be applied for real sample analysis.

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4. Conclusion Herein, an efficient electrochemical sensor was developed for lead ion detection using a DNAzyme cleavage-induced templateindependent polymerization and alkaline phosphatase amplification strategy. It took fully advantages of triply cascade signal amplification effects including cyclic Pb2 þ -dependent DNAzyme cleavage, TdTase-mediated base extension and enzymatic catalysis of ALP, which guaranteed the ultrasensitive detection of Pb2 þ with an impressive low detection limit of about 0.043 nM. It also exhibited the distinct advantages of simplicity or flexibility in probe design and biosensor fabrication. The relatively long assay time for the current strategy might be improved by engineering the catalytic strand and substrate strand of 8–17 DNAzyme as a unimolecular DNA probe to avoid the hybridization process. Also, the screening of suitable experimental conditions to install the onestep operation for the Pb2 þ -dependent cleavage reaction and TdTase-mediated base extension might further shorten the assay time for Pb2 þ . Another limitation of this sensing device is the single use of the sensing surface. However, the proposed strategy should be easily extended to analyze other metal ions using the respective metal ion-dependent DNAzymes or some nucleic acidrelated analytes and thus it opens a promising avenue for the development of electrochemical sensors.

Acknowledgement This work was funded by the National Natural Science Foundation of China (No. 21475072) and the Natural Science Foundation of Shandong Province of China (Nos. ZR2015JL007, ZR2014BM019).

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2016.04.026.

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