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A novel signal-on electrochemical DNA sensor based on target catalyzed hairpin assembly strategy
Biosensors and Bioelectronics 64 (2015) 177–181
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short Communication
A novel signal-on electrochemical DNA sensor based on target catalyzed hairpin assembly strategy Yong Qian a, Daoquan Tang b, Lili Du b, Yanzhuo Zhang b, Lixian Zhang b, Fenglei Gao b,n a b
Fundamental Science on Radioactive Geology and Exploration Technology Laboratory, East China Institute of Technology, Nanchang, Jiangxi 330013, China Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy, Xuzhou Medical College, 221004 Xuzhou, China
art ic l e i nf o
a b s t r a c t
Article history: Received 25 June 2014 Received in revised form 21 August 2014 Accepted 1 September 2014 Available online 6 September 2014
We describe a novel signal-on electrochemical DNA (E-DNA) sensing platform based on target-catalyzed hairpin assembly. The thiolated modified molecular beacon 1 (MB1) was first immobilized onto the Au electrode (GE) surface and then target DNA hybridized to the MB1, the opened MB1 assembled with the ferrocene (Fc)-labeled molecular beacon 2 to displace the target DNA, which became available for the next cycle of MB1 target hybridization. Moreover, Fc was confined close to the GE surface for efficient electron transfer, resulting in a current signal. Eventually, each target strand went through many cycles, resulting in numerous Fcs confining close to the GE, which leaded to the current of Fc dramatically increase. The observed signal gain was sufficient to achieve a demonstrated detection limit of 0.74 fM, with a wide linear dynamic range from 10 15 to 10 10 M and discriminated mismatched DNA from perfect matched target DNA with a high selectivity. Thus, the proposed E-DNA sensor would have a wide range of sensor applications because it is enzyme-free and simple to perform. & 2014 Elsevier B.V. All rights reserved.
Keywords: Signal on Molecular beacon Electrochemical Sensors
1. Introduction DNA detection is great demand in gene profiling, drug screening, clinical diagnostics, environmental analysis, and food safety (Drummond et al., 2003; Guo et al., 2009; Chen et al., 2011; Liu et al., 2011). Motivated by this demand, various techniques for DNA detection have been developed, such as electrochemical (Fan et al., 2005; Hu et al., 2012; Tang et al., 2013), fluorescent (Zhao et al., 2012), chemiluminescent (Liu et al., 2013) methods. Up to now, electrochemical biosensors have received great interests because of the simplicity, fast response, relatively cheap cost, high sensitivity, and low power requirement of electrochemical methods (Zhang et al., 2013). Recent years, we have seen the development of a number of electrochemical DNA (E-DNA) sensors that detect hybridization-induced conformational changes in a redox tags (e.g. ferrocene or methylene blue)-modified, electrode-bound probe DNA, such as “signal-on” and “signal-off” E-DNA architectures (Fan et al., 2003; Immoos et al., 2004; Xiao et al., 2007; Wu and Lai, 2013). These E-DNA sensors require no addition of reagents or target labeling, and detection is a rapid single-step process. Additionally, as the signaling mechanism is linked to a specific conformational change, these sensors are capable of n
Corresponding author. Fax: þ 86 516 83262138. E-mail address: jsxzgfl@sina.com (F. Gao).
http://dx.doi.org/10.1016/j.bios.2014.09.001 0956-5663/& 2014 Elsevier B.V. All rights reserved.
functioning in complex, multicomponent samples (Lubin et al., 2006). In a “signal-off” sensor, the mechanism is the alternation of distance of the labeled redox tags from the electrode by target DNA-induced conformational change of probe (Lubin and Plaxco, 2010; Wang et al., 2012). However, such “signal-off” sensors suffer from limited signal capacity, in which only a maximum of 100% signal suppression can be attained under any experimental conditions (Ricci et al., 2007). Moreover, such “signal-off” assays might cause false-positive results due to the coexistence of environmental stimulus (Anne et al., 2003; Lubin et al., 2006). To circumvent this limitation, “signal-on” E-DNA sensors have been developed in the past years. In contrast, “signal-on” sensors can achieve much improved signaling, and the background current observed in the absence of target is reduced, the gain of such a sensor, at least in theory, increases without limit. Thus motivated, others have explored a number of “signal-on” E-DNA architectures, such as DNA pseudoknot (Cash et al., 2009), hybridization-based double-stranded (Xiao et al., 2006), triblock structure (Immoos et al., 2004), inverted stem-loop (Rowe et al., 2011), triplex DNA structure (Idili et al., 2014), and traditional E-DNA sensor probed at new frequencies (White and Plaxco, 2010). Despite these advances, above-mentioned “signal-on” strategy use redox-labeled capture DNA strand to provide a response signal of target. Unfortunately, although the labeled redox tags of capture DNA far away the electrode, they still have a high background
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Y. Qian et al. / Biosensors and Bioelectronics 64 (2015) 177–181
Scheme 1. Schematic of the signal-on E-DNA sensor, which is based on target catalyzing hairpin assembly strategy.
signal. Furmore, above-mentioned “signal-on” sensor is not sensitive enough, because a single target DNA molecule only reacts with a single signaling probe, limiting the total signal gain. In this paper, we reported an efficient “signal-on” electrochemical DNA sensing system based on target catalyzed hairpin assembly, which do not produce a background signal. The target catalyzed hairpin assembly has recently attracted considerable attention due to enzyme-free DNA detection, in which the signal amplification is achieved by the cycling use of the target (Allen et al., 2012; Huang et al., 2012; Zheng et al., 2012). As shown in Scheme 1, the thiolated modified molecular beacon 1 (MB1) was first immobilized on the Au electrode (GE) surface and then target DNA hybridizes to the MB1, the opened MB1 assembles with the ferrocene (Fc)-labeled molecular beacon 2 (MB2) to displace the target DNA, which becomes available for the next cycle of MB1 target hybridization. Moreover, Fc is confined close to the electrode surface for efficient electron transfer, resulting in a large current of the redox. Eventually, each target strand can go through many cycles, resulting in many Fc confining close to the GE which leads to the current of Fc dramatically enhancement. By monitoring the change of the current intensity, we can detect the target DNA with high sensitivity. In the absence of target, the MB1 and MB2 can only maintain the sufficiently stable stem-loop structure owing to the binding of the complementary sequences at the ends. Therefore, in addition to the simple and relatively lowcost detection process, the employment of MBs and the recycle of the target make this strategy appealing in amplifying electrochemical signal for selective and sensitive DNA detection.
2. Experimental 2.1. Oligonucleotides and regents Water was purified with a Milli-Q purification system (Branstead, USA) and used throughout the work. All chemicals used in this work were of analytical grade. The buffers used in the study were HEPES buffer (10 mM HEPES,150 mM NaCl, pH 7.4) for target binding, The washing buffer was PBS (50 mM Na2HPO4, 50 mM NaH2PO4, 1 M NaCl, pH 7.5). To avoid the instability of ferrocenium (the oxidized form of the ferrocene), 1.0 M NaClO4 solution was used as the supporting electrolyte when electrochemical behavior of the working electrode was investigated. DNA oligonucleotides used in this work were synthesized and purified by Takara Biotechnology Co., Ltd. (Dalian, China). MB1:5′-SH-AAGTAGTGATTGAGCGTGATGAATGTCACTAC TTCAACTCGCATTCATCACGCTCAATC-3′ MB2:5′-TGATGAATGCGAGTTGAAGTAGTGACATTCA TCACGCTCAATCACTACTTCAACTCGCA-Fc-3′ Target:5′-GACATTCATCACGCTCAATCACTACTT-3′ Single-base mismatched:5′-GACATTCATCAC ACTCAATCACTACTT-3′
Three-base mismatched:5′-GACATTCATCACAC TCAATCACTACTT-3′ Non-complementary:5′-ATGCTGACTGACAAG CTTAGCAAGGG-3′
2.2. Electrode modification Prior to modification, the bare GE (3 mm in diameter) was polished to a mirror-like surface with alumina suspensions and then sequentially cleaned ultrasonically in 95% ethanol and twice-quartz-distilled water for 5 min. Prior to attachment to the GE surface, 100 μL of 100 μM thiolated MB1 was incubated with 0.1 μL of 100 mM TCEP for 1 h to reduce disulfide bonds and subsequently diluted to 1.0 μM with phosphate buffer. 10 μL of thiolated MB1 (1 μM) was dropped on the cleaned GE for 4 h at room temperature in the dark. During this process, the MB1 was conjugated onto the GE via the Au‒S bond. After rinsing with distilled water, the modified GE was incubated with 1.0 mM 6-mercaptohexanol in10 mM Tris–HCl buffer (pH7.4) for 1 h at room temperature. Finally, 5 μL target DNA with the designed concentration and 5 μL MB2 (300 nM) were dropped on the surface of the electrodes. After the process was performed for 2 h at 37 °C, it was terminated by washing thoroughly. The whole procedure was shown in Scheme 1. 2.3. Measurement procedure Electrochemical experiments were carried out using the CHI 660C electrochemical analyzer. Cyclic voltammetry (CV) results were recorded within a potential range of 0–0.6 V (scan rate¼0.05 V s 1). For all measurements, 4 successive cycles were carried out to ensure signal stabilization and the fourth cycle was kept as the result. Differential pulse voltammograms (DPVs) of Fc tag were registered in the potential interval 0.0 to þ0.6 V vs. Ag/AgCl under the following conditions: modulation amplitude 0.05 V, pulse width 0.06 s, and sample width 0.02 s. The Electrochemical impedance spectroscopy (EIS) measurement was also carried out with the CHI 660C electrochemical analyzer. Supporting electrolyte solution was 1.0 mmol/L K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) solution containing 0.1 mol/L KCl. The ac voltage amplitude was 5 mV, and the voltage frequencies used for EIS measurements ranged from 100 kHz to 100 mHz. The applied potential was 172 mV vs. Ag/AgCl (Zhang et al., 2009). This potential is near the equilibrium of [Fe(CN)6]3 /4 pair, and makes the redox rates equal. Therefore, the redox species will not be depleted near the electrode surface during the measurement (Pan and Rothberg, 2005).
3. Results and discussion 3.1. Characterization of the E-DNA biosensor CV measurements were used to monitor the change in surface features of the electrode after each step modification in this work.
Y. Qian et al. / Biosensors and Bioelectronics 64 (2015) 177–181
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Fig. 1. (A) CV curves of (a) the electrode immobilized with MB1, (b) after the hybridization of MB1 and 1.0 pM target DNA sequences, (c) 1.0 pM target DNA þ 0.3 μM MB2, and (d) 0.1 nM target DNA þ 0.3 μM MB2. The electrolyte is 1 M NaClO4. (B) EIS spectroscopy during the progress of (a) bare gold electrode, (b) the electrode immobilized with MB1; (c) after the hybridization of MB1 and target sequences; (d) after the hybridization of MB1 and target sequences in the presence of MB2 for 2 h. (C) DPVs curves for the sensor in the presence of (a) blank, (b) 0.1 nM target, (c) 0.3 μM MB2, (d) 0.1 nM target, and 0.3 μM MB2 for 0.5 h, (e) 0.1 nM target, and 0.3 μM MB2 for 2 h.
As shown in Fig. 1A, no redox couple is seen in this potential window for electrodes modified with MB1 in the absence of target DNA (curve a). When Fc-labled MB2 was hybridized with MB1 modified on the electrode in the presence of target DNA, a couple of strong redox peaks appeared at 0.241 and 0.294 V in CV (curve b), and indicating electrochemical characteristics of Fc confining close to electrode surface. The redox response changes significantly in the presence of high concentration target DNA. As shown in curve c, addition of 0.1 nM target DNA and MB2 to the GE surface causes an increase in current associated with the Fc probe. These observations indicated that the redox process took place only from the surface and this biosensor was sensitively responsive to target DN A (Fan et al., 2003, Chatelain et al., 2012). The stepwise modifications of the sensing electrode surface were monitored by EIS. In a typical EIS spectrum, the diameter of the semicircle reveals the electron transfer resistance (Ret) (Cheng et al., 2014). As shown in Fig. 1B, the bare gold electrode shows a very small semicircle domain (curve a), indicating a very fast charge-transfer process. When the thiolated capture MB1 was selfassembled onto the bare electrode via Au–thiol binding, the Ret increased (curve b). This was because that the negatively charged phosphate backbone of the oligonucleotides produced an electrostatic repulsion force to [Fe(CN)6]3 /[Fe(CN)6]4 (Wang et al., 2013). After hybridization with target DNA, Ret further increases (curve c), suggesting the formation of the ds-DNA on the electrode surface. The Ret largely increased when MB2 were attached to the GE surface due to the cycling use of the target DNA and the
continuous generation many of the MB1 MB2 complex (curve d). All these experimental results demonstrate the sensing interface has been successfully fabricated according to Scheme 1.
3.2. Feasibility of the E-DNA biosensor To further verify the mechanism of the assay, DPV responses of the different modified electrodes were investigated. As shown in Fig. 1C, no current was produced in the only presence of a complementary target (curve b) which the same with blank (curve a), because no Fc was introduced to the surface of GE. Furthermore, in the MB2 assisted system, after hybridization with Fc– MB2, the sensor produces a sharply current peak of the Fc response current at about 0.294 V (curve d) and increased with the reaction time (curve e). Clearly, the signal enhancement was caused by the cycling use of the target DNA and the continuous generation of the MB1 MB2 complex as shown in Fig. 1C, which allowing the Fc to approach the electrode surface and transfer electrons. In addition, for the solution containing MB2 only, no current intensity was observed (curve c), indicating that the hairpin structures of MB2 could not open the loop of MB1 owing to the binding of the complementary sequences at the ends. These results clearly indicated that the developed E-DNA biosensor by coupling “signal-on”strategies could pave a new way to detect target DNA.
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The target catalyzed hairpin assembly process could be affected by two factors including the MB2 concentration and the incubation time. In order to achieve the best signal-to-noise level, the reaction conditions were optimized. To investigate the effect of the concentration of MB2 on the detection system, target DNA (10 fM) was mixed with MB2 at different concentrations ranging from 0 to 350 nM. As shown in Fig. S1A, the current intensity of the system increased as the concentration of MB2 increased. Furthermore, the optimum concentration of MB2 used in this system was 300 nM due to its best signal-to-noise level. As shown in Fig. S1B, it could be found that the electrochemical signal increased with the reaction time before reaching saturation after 2 h, indicating strongly that target DNA could go through many cycles to open the MB1. Considering this issue, 2 h was chosen as the reaction time. It is seen that the signal of the sensing system increased with increase of the concentration of MB2 and time in the absence of the target DNA. This background signal was due to the hybridization of MB1 with MB2. Fortunately, the increment was so small, and could be ignored. Upon the addition of the target DNA, the current signal of the sensing system was significantly larger than the control.
approach the electrode surface to generate strong current. On the other hand, do not produce a background signal because the hairpin structures of MB2 could not open the loop of MB1. The result suggested that this sensing system was appropriate for quantitative determination of target DNA concentration. In this study, we evaluate the specificity of this method. The assay is challenged with 0.1 pM single-base mismatched, threemismatched, non-complementary and complementary target DNA. As indicated from Fig. 2B, higher current was observed with complementary target DNA than those of other mismatched DNA sequences. Although the current intensity increased with the increasing concentration of the mismatched DNA (Fig. S2), the current signals for mismatched DNA were much lower than those for the perfectly matched target at the same concentration, and even 50 nmol L 1 single-base mismatched DNA showed a signal lower than that of the perfectly matched target at 1.0 fmol L 1. So these results demonstrated that this DNA sensing system can be used to discriminate perfectly matched and mismatched DNA targets. This is because the relatively long stem of the MBs makes the hairpin structure thermodynamically stable, and it is unfavorable for the hybridization between mismatched sequences and the MBs.
3.4. Analytical performance of the biosensor
3.5. Reproducibility and stability of the DNA biosensor
Under the best optimal experimental conditions, we examined the sensitivity of the proposed method upon the addition of different concentrations of the target DNA. As shown in Fig. 2A, the DPV peak current increased with the increasing of DNA concentration. The calibration plots displayed a good linear relationship between the peak currents and the logarithm of DNA concentrations in the range from 1.0 fM to 0.1 nM (inset of Fig. 2A) and the fitted regression equation is I ¼0.022 Log c þ0.342 (R2 ¼0.9955) (I is the peak current (μA) and c is the concentration of the target DNA (mol L 1)) with a low detection limit of 0.74 fM from three times the standard deviation corresponding to the blank sample detection, providing superior detection sensitivity compared with those reported “signal-on” electrochemical methods including a hybridization-based double-stranded sensor (400 fM) (Xiao et al., 2006), a triblock structure (200 pM) (Immoos et al., 2004), triplex DNA structure (10 pM) (Idili et al., 2014) and an inverted stem-loop (50 pM) (Rowe et al., 2011). The low detection limit of this method is attributed to the mechanism of the target DNA catalyzed hairpin assembly strategies, on one hand, which makes the target DNA recycle for allowing many Fc to
The reproducibility of the electrode was also investigated. Five modified electrodes were used to detect target DNA (0.1 pM), and the relative standard deviation is 5.2%. Furthermore, the sensor remains bioactive after two-week storage at 4 °C, and the response currents have no significant changes. The result demonstrates that the developed E-DNA biosensor has satisfactory reproducibility and stability.
3.3. Optimization of experimental conditions
3.6. Real samples assay To investigate the validity of the proposed signal amplification strategy to real clinical samples, we challenged our sensing strategy with the presence of the target DNA in a complex sample matrix, 10% human serum (diluted with buffer). Two target DNA samples including 1.0 pM and 0.1 nM were spiked into the human serum, which were assayed by the developed DNA sensors. The recoveries were 95.3% and 103.4%, respectively, which demonstrated the capacity of this assay in the analysis of real samples where the influence of the matrix could be neglected.
Fig. 2. (A) DPV curves of Fc for detection of DNA at target DNA concentrations of 10 16–10 9 mol L 1 (a–h) in the presence of MB2; Inset: Linear relationship between peak current and the logarithm of target DNA concentration. (B) Histograms of current intensity for (a) blank, (b) 0.1 pmol L 1 non-complementary, (c) three-base mismatched, (d) single-base mismatched, and (e) complementary target DNA. (n¼3)
Y. Qian et al. / Biosensors and Bioelectronics 64 (2015) 177–181
4. Conclusions Here we have demonstrated a simple “signal-on” electrochemical DNA sensor based on target catalyzed hairpin assembly. The employment of two different types of MBs, on one hand, makes the target available for inducing circular reactions, which allowing many Fc to approach the electrode surface for generating strong current. On the other hand, do not produce a background signal. The detection limit of this method was 0.74 fM, which are about 3 orders of magnitude lower than that of the conventional “signal-on” methods without amplification mechanisms. Another advantage of the method is protein enzymes-free and thermal cycling procedures-free, which make this method inexpensive and simple. Furthermore, the special structures of the MBs, such as the long stem, promote the detection selectivity. The high sensitivity and selectivity make this method a great potential for early diagnosis in gene-related diseases. We believe that our new analytical method will have promising applications in the sensitive and selective electrochemical determination of other small molecules and proteins.
Acknowledgment This work was supported by the National Natural Science Foundation of China (21405130, 21264001), Excellent Talents of Xuzhou Medical College (D2014007), Natural Science Foundation of Jiangxi Province (20114BAB213010) and Young Scientist Foundation of Jiangxi Province (20133BCB23020).
Appendix A. Supplementary Information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.09.001.
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Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short Communication
A novel signal-on electrochemical DNA sensor based on target catalyzed hairpin assembly strategy Yong Qian a, Daoquan Tang b, Lili Du b, Yanzhuo Zhang b, Lixian Zhang b, Fenglei Gao b,n a b
Fundamental Science on Radioactive Geology and Exploration Technology Laboratory, East China Institute of Technology, Nanchang, Jiangxi 330013, China Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy, Xuzhou Medical College, 221004 Xuzhou, China
art ic l e i nf o
a b s t r a c t
Article history: Received 25 June 2014 Received in revised form 21 August 2014 Accepted 1 September 2014 Available online 6 September 2014
We describe a novel signal-on electrochemical DNA (E-DNA) sensing platform based on target-catalyzed hairpin assembly. The thiolated modified molecular beacon 1 (MB1) was first immobilized onto the Au electrode (GE) surface and then target DNA hybridized to the MB1, the opened MB1 assembled with the ferrocene (Fc)-labeled molecular beacon 2 to displace the target DNA, which became available for the next cycle of MB1 target hybridization. Moreover, Fc was confined close to the GE surface for efficient electron transfer, resulting in a current signal. Eventually, each target strand went through many cycles, resulting in numerous Fcs confining close to the GE, which leaded to the current of Fc dramatically increase. The observed signal gain was sufficient to achieve a demonstrated detection limit of 0.74 fM, with a wide linear dynamic range from 10 15 to 10 10 M and discriminated mismatched DNA from perfect matched target DNA with a high selectivity. Thus, the proposed E-DNA sensor would have a wide range of sensor applications because it is enzyme-free and simple to perform. & 2014 Elsevier B.V. All rights reserved.
Keywords: Signal on Molecular beacon Electrochemical Sensors
1. Introduction DNA detection is great demand in gene profiling, drug screening, clinical diagnostics, environmental analysis, and food safety (Drummond et al., 2003; Guo et al., 2009; Chen et al., 2011; Liu et al., 2011). Motivated by this demand, various techniques for DNA detection have been developed, such as electrochemical (Fan et al., 2005; Hu et al., 2012; Tang et al., 2013), fluorescent (Zhao et al., 2012), chemiluminescent (Liu et al., 2013) methods. Up to now, electrochemical biosensors have received great interests because of the simplicity, fast response, relatively cheap cost, high sensitivity, and low power requirement of electrochemical methods (Zhang et al., 2013). Recent years, we have seen the development of a number of electrochemical DNA (E-DNA) sensors that detect hybridization-induced conformational changes in a redox tags (e.g. ferrocene or methylene blue)-modified, electrode-bound probe DNA, such as “signal-on” and “signal-off” E-DNA architectures (Fan et al., 2003; Immoos et al., 2004; Xiao et al., 2007; Wu and Lai, 2013). These E-DNA sensors require no addition of reagents or target labeling, and detection is a rapid single-step process. Additionally, as the signaling mechanism is linked to a specific conformational change, these sensors are capable of n
Corresponding author. Fax: þ 86 516 83262138. E-mail address: jsxzgfl@sina.com (F. Gao).
http://dx.doi.org/10.1016/j.bios.2014.09.001 0956-5663/& 2014 Elsevier B.V. All rights reserved.
functioning in complex, multicomponent samples (Lubin et al., 2006). In a “signal-off” sensor, the mechanism is the alternation of distance of the labeled redox tags from the electrode by target DNA-induced conformational change of probe (Lubin and Plaxco, 2010; Wang et al., 2012). However, such “signal-off” sensors suffer from limited signal capacity, in which only a maximum of 100% signal suppression can be attained under any experimental conditions (Ricci et al., 2007). Moreover, such “signal-off” assays might cause false-positive results due to the coexistence of environmental stimulus (Anne et al., 2003; Lubin et al., 2006). To circumvent this limitation, “signal-on” E-DNA sensors have been developed in the past years. In contrast, “signal-on” sensors can achieve much improved signaling, and the background current observed in the absence of target is reduced, the gain of such a sensor, at least in theory, increases without limit. Thus motivated, others have explored a number of “signal-on” E-DNA architectures, such as DNA pseudoknot (Cash et al., 2009), hybridization-based double-stranded (Xiao et al., 2006), triblock structure (Immoos et al., 2004), inverted stem-loop (Rowe et al., 2011), triplex DNA structure (Idili et al., 2014), and traditional E-DNA sensor probed at new frequencies (White and Plaxco, 2010). Despite these advances, above-mentioned “signal-on” strategy use redox-labeled capture DNA strand to provide a response signal of target. Unfortunately, although the labeled redox tags of capture DNA far away the electrode, they still have a high background
178
Y. Qian et al. / Biosensors and Bioelectronics 64 (2015) 177–181
Scheme 1. Schematic of the signal-on E-DNA sensor, which is based on target catalyzing hairpin assembly strategy.
signal. Furmore, above-mentioned “signal-on” sensor is not sensitive enough, because a single target DNA molecule only reacts with a single signaling probe, limiting the total signal gain. In this paper, we reported an efficient “signal-on” electrochemical DNA sensing system based on target catalyzed hairpin assembly, which do not produce a background signal. The target catalyzed hairpin assembly has recently attracted considerable attention due to enzyme-free DNA detection, in which the signal amplification is achieved by the cycling use of the target (Allen et al., 2012; Huang et al., 2012; Zheng et al., 2012). As shown in Scheme 1, the thiolated modified molecular beacon 1 (MB1) was first immobilized on the Au electrode (GE) surface and then target DNA hybridizes to the MB1, the opened MB1 assembles with the ferrocene (Fc)-labeled molecular beacon 2 (MB2) to displace the target DNA, which becomes available for the next cycle of MB1 target hybridization. Moreover, Fc is confined close to the electrode surface for efficient electron transfer, resulting in a large current of the redox. Eventually, each target strand can go through many cycles, resulting in many Fc confining close to the GE which leads to the current of Fc dramatically enhancement. By monitoring the change of the current intensity, we can detect the target DNA with high sensitivity. In the absence of target, the MB1 and MB2 can only maintain the sufficiently stable stem-loop structure owing to the binding of the complementary sequences at the ends. Therefore, in addition to the simple and relatively lowcost detection process, the employment of MBs and the recycle of the target make this strategy appealing in amplifying electrochemical signal for selective and sensitive DNA detection.
2. Experimental 2.1. Oligonucleotides and regents Water was purified with a Milli-Q purification system (Branstead, USA) and used throughout the work. All chemicals used in this work were of analytical grade. The buffers used in the study were HEPES buffer (10 mM HEPES,150 mM NaCl, pH 7.4) for target binding, The washing buffer was PBS (50 mM Na2HPO4, 50 mM NaH2PO4, 1 M NaCl, pH 7.5). To avoid the instability of ferrocenium (the oxidized form of the ferrocene), 1.0 M NaClO4 solution was used as the supporting electrolyte when electrochemical behavior of the working electrode was investigated. DNA oligonucleotides used in this work were synthesized and purified by Takara Biotechnology Co., Ltd. (Dalian, China). MB1:5′-SH-AAGTAGTGATTGAGCGTGATGAATGTCACTAC TTCAACTCGCATTCATCACGCTCAATC-3′ MB2:5′-TGATGAATGCGAGTTGAAGTAGTGACATTCA TCACGCTCAATCACTACTTCAACTCGCA-Fc-3′ Target:5′-GACATTCATCACGCTCAATCACTACTT-3′ Single-base mismatched:5′-GACATTCATCAC ACTCAATCACTACTT-3′
Three-base mismatched:5′-GACATTCATCACAC TCAATCACTACTT-3′ Non-complementary:5′-ATGCTGACTGACAAG CTTAGCAAGGG-3′
2.2. Electrode modification Prior to modification, the bare GE (3 mm in diameter) was polished to a mirror-like surface with alumina suspensions and then sequentially cleaned ultrasonically in 95% ethanol and twice-quartz-distilled water for 5 min. Prior to attachment to the GE surface, 100 μL of 100 μM thiolated MB1 was incubated with 0.1 μL of 100 mM TCEP for 1 h to reduce disulfide bonds and subsequently diluted to 1.0 μM with phosphate buffer. 10 μL of thiolated MB1 (1 μM) was dropped on the cleaned GE for 4 h at room temperature in the dark. During this process, the MB1 was conjugated onto the GE via the Au‒S bond. After rinsing with distilled water, the modified GE was incubated with 1.0 mM 6-mercaptohexanol in10 mM Tris–HCl buffer (pH7.4) for 1 h at room temperature. Finally, 5 μL target DNA with the designed concentration and 5 μL MB2 (300 nM) were dropped on the surface of the electrodes. After the process was performed for 2 h at 37 °C, it was terminated by washing thoroughly. The whole procedure was shown in Scheme 1. 2.3. Measurement procedure Electrochemical experiments were carried out using the CHI 660C electrochemical analyzer. Cyclic voltammetry (CV) results were recorded within a potential range of 0–0.6 V (scan rate¼0.05 V s 1). For all measurements, 4 successive cycles were carried out to ensure signal stabilization and the fourth cycle was kept as the result. Differential pulse voltammograms (DPVs) of Fc tag were registered in the potential interval 0.0 to þ0.6 V vs. Ag/AgCl under the following conditions: modulation amplitude 0.05 V, pulse width 0.06 s, and sample width 0.02 s. The Electrochemical impedance spectroscopy (EIS) measurement was also carried out with the CHI 660C electrochemical analyzer. Supporting electrolyte solution was 1.0 mmol/L K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) solution containing 0.1 mol/L KCl. The ac voltage amplitude was 5 mV, and the voltage frequencies used for EIS measurements ranged from 100 kHz to 100 mHz. The applied potential was 172 mV vs. Ag/AgCl (Zhang et al., 2009). This potential is near the equilibrium of [Fe(CN)6]3 /4 pair, and makes the redox rates equal. Therefore, the redox species will not be depleted near the electrode surface during the measurement (Pan and Rothberg, 2005).
3. Results and discussion 3.1. Characterization of the E-DNA biosensor CV measurements were used to monitor the change in surface features of the electrode after each step modification in this work.
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Fig. 1. (A) CV curves of (a) the electrode immobilized with MB1, (b) after the hybridization of MB1 and 1.0 pM target DNA sequences, (c) 1.0 pM target DNA þ 0.3 μM MB2, and (d) 0.1 nM target DNA þ 0.3 μM MB2. The electrolyte is 1 M NaClO4. (B) EIS spectroscopy during the progress of (a) bare gold electrode, (b) the electrode immobilized with MB1; (c) after the hybridization of MB1 and target sequences; (d) after the hybridization of MB1 and target sequences in the presence of MB2 for 2 h. (C) DPVs curves for the sensor in the presence of (a) blank, (b) 0.1 nM target, (c) 0.3 μM MB2, (d) 0.1 nM target, and 0.3 μM MB2 for 0.5 h, (e) 0.1 nM target, and 0.3 μM MB2 for 2 h.
As shown in Fig. 1A, no redox couple is seen in this potential window for electrodes modified with MB1 in the absence of target DNA (curve a). When Fc-labled MB2 was hybridized with MB1 modified on the electrode in the presence of target DNA, a couple of strong redox peaks appeared at 0.241 and 0.294 V in CV (curve b), and indicating electrochemical characteristics of Fc confining close to electrode surface. The redox response changes significantly in the presence of high concentration target DNA. As shown in curve c, addition of 0.1 nM target DNA and MB2 to the GE surface causes an increase in current associated with the Fc probe. These observations indicated that the redox process took place only from the surface and this biosensor was sensitively responsive to target DN A (Fan et al., 2003, Chatelain et al., 2012). The stepwise modifications of the sensing electrode surface were monitored by EIS. In a typical EIS spectrum, the diameter of the semicircle reveals the electron transfer resistance (Ret) (Cheng et al., 2014). As shown in Fig. 1B, the bare gold electrode shows a very small semicircle domain (curve a), indicating a very fast charge-transfer process. When the thiolated capture MB1 was selfassembled onto the bare electrode via Au–thiol binding, the Ret increased (curve b). This was because that the negatively charged phosphate backbone of the oligonucleotides produced an electrostatic repulsion force to [Fe(CN)6]3 /[Fe(CN)6]4 (Wang et al., 2013). After hybridization with target DNA, Ret further increases (curve c), suggesting the formation of the ds-DNA on the electrode surface. The Ret largely increased when MB2 were attached to the GE surface due to the cycling use of the target DNA and the
continuous generation many of the MB1 MB2 complex (curve d). All these experimental results demonstrate the sensing interface has been successfully fabricated according to Scheme 1.
3.2. Feasibility of the E-DNA biosensor To further verify the mechanism of the assay, DPV responses of the different modified electrodes were investigated. As shown in Fig. 1C, no current was produced in the only presence of a complementary target (curve b) which the same with blank (curve a), because no Fc was introduced to the surface of GE. Furthermore, in the MB2 assisted system, after hybridization with Fc– MB2, the sensor produces a sharply current peak of the Fc response current at about 0.294 V (curve d) and increased with the reaction time (curve e). Clearly, the signal enhancement was caused by the cycling use of the target DNA and the continuous generation of the MB1 MB2 complex as shown in Fig. 1C, which allowing the Fc to approach the electrode surface and transfer electrons. In addition, for the solution containing MB2 only, no current intensity was observed (curve c), indicating that the hairpin structures of MB2 could not open the loop of MB1 owing to the binding of the complementary sequences at the ends. These results clearly indicated that the developed E-DNA biosensor by coupling “signal-on”strategies could pave a new way to detect target DNA.
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The target catalyzed hairpin assembly process could be affected by two factors including the MB2 concentration and the incubation time. In order to achieve the best signal-to-noise level, the reaction conditions were optimized. To investigate the effect of the concentration of MB2 on the detection system, target DNA (10 fM) was mixed with MB2 at different concentrations ranging from 0 to 350 nM. As shown in Fig. S1A, the current intensity of the system increased as the concentration of MB2 increased. Furthermore, the optimum concentration of MB2 used in this system was 300 nM due to its best signal-to-noise level. As shown in Fig. S1B, it could be found that the electrochemical signal increased with the reaction time before reaching saturation after 2 h, indicating strongly that target DNA could go through many cycles to open the MB1. Considering this issue, 2 h was chosen as the reaction time. It is seen that the signal of the sensing system increased with increase of the concentration of MB2 and time in the absence of the target DNA. This background signal was due to the hybridization of MB1 with MB2. Fortunately, the increment was so small, and could be ignored. Upon the addition of the target DNA, the current signal of the sensing system was significantly larger than the control.
approach the electrode surface to generate strong current. On the other hand, do not produce a background signal because the hairpin structures of MB2 could not open the loop of MB1. The result suggested that this sensing system was appropriate for quantitative determination of target DNA concentration. In this study, we evaluate the specificity of this method. The assay is challenged with 0.1 pM single-base mismatched, threemismatched, non-complementary and complementary target DNA. As indicated from Fig. 2B, higher current was observed with complementary target DNA than those of other mismatched DNA sequences. Although the current intensity increased with the increasing concentration of the mismatched DNA (Fig. S2), the current signals for mismatched DNA were much lower than those for the perfectly matched target at the same concentration, and even 50 nmol L 1 single-base mismatched DNA showed a signal lower than that of the perfectly matched target at 1.0 fmol L 1. So these results demonstrated that this DNA sensing system can be used to discriminate perfectly matched and mismatched DNA targets. This is because the relatively long stem of the MBs makes the hairpin structure thermodynamically stable, and it is unfavorable for the hybridization between mismatched sequences and the MBs.
3.4. Analytical performance of the biosensor
3.5. Reproducibility and stability of the DNA biosensor
Under the best optimal experimental conditions, we examined the sensitivity of the proposed method upon the addition of different concentrations of the target DNA. As shown in Fig. 2A, the DPV peak current increased with the increasing of DNA concentration. The calibration plots displayed a good linear relationship between the peak currents and the logarithm of DNA concentrations in the range from 1.0 fM to 0.1 nM (inset of Fig. 2A) and the fitted regression equation is I ¼0.022 Log c þ0.342 (R2 ¼0.9955) (I is the peak current (μA) and c is the concentration of the target DNA (mol L 1)) with a low detection limit of 0.74 fM from three times the standard deviation corresponding to the blank sample detection, providing superior detection sensitivity compared with those reported “signal-on” electrochemical methods including a hybridization-based double-stranded sensor (400 fM) (Xiao et al., 2006), a triblock structure (200 pM) (Immoos et al., 2004), triplex DNA structure (10 pM) (Idili et al., 2014) and an inverted stem-loop (50 pM) (Rowe et al., 2011). The low detection limit of this method is attributed to the mechanism of the target DNA catalyzed hairpin assembly strategies, on one hand, which makes the target DNA recycle for allowing many Fc to
The reproducibility of the electrode was also investigated. Five modified electrodes were used to detect target DNA (0.1 pM), and the relative standard deviation is 5.2%. Furthermore, the sensor remains bioactive after two-week storage at 4 °C, and the response currents have no significant changes. The result demonstrates that the developed E-DNA biosensor has satisfactory reproducibility and stability.
3.3. Optimization of experimental conditions
3.6. Real samples assay To investigate the validity of the proposed signal amplification strategy to real clinical samples, we challenged our sensing strategy with the presence of the target DNA in a complex sample matrix, 10% human serum (diluted with buffer). Two target DNA samples including 1.0 pM and 0.1 nM were spiked into the human serum, which were assayed by the developed DNA sensors. The recoveries were 95.3% and 103.4%, respectively, which demonstrated the capacity of this assay in the analysis of real samples where the influence of the matrix could be neglected.
Fig. 2. (A) DPV curves of Fc for detection of DNA at target DNA concentrations of 10 16–10 9 mol L 1 (a–h) in the presence of MB2; Inset: Linear relationship between peak current and the logarithm of target DNA concentration. (B) Histograms of current intensity for (a) blank, (b) 0.1 pmol L 1 non-complementary, (c) three-base mismatched, (d) single-base mismatched, and (e) complementary target DNA. (n¼3)
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4. Conclusions Here we have demonstrated a simple “signal-on” electrochemical DNA sensor based on target catalyzed hairpin assembly. The employment of two different types of MBs, on one hand, makes the target available for inducing circular reactions, which allowing many Fc to approach the electrode surface for generating strong current. On the other hand, do not produce a background signal. The detection limit of this method was 0.74 fM, which are about 3 orders of magnitude lower than that of the conventional “signal-on” methods without amplification mechanisms. Another advantage of the method is protein enzymes-free and thermal cycling procedures-free, which make this method inexpensive and simple. Furthermore, the special structures of the MBs, such as the long stem, promote the detection selectivity. The high sensitivity and selectivity make this method a great potential for early diagnosis in gene-related diseases. We believe that our new analytical method will have promising applications in the sensitive and selective electrochemical determination of other small molecules and proteins.
Acknowledgment This work was supported by the National Natural Science Foundation of China (21405130, 21264001), Excellent Talents of Xuzhou Medical College (D2014007), Natural Science Foundation of Jiangxi Province (20114BAB213010) and Young Scientist Foundation of Jiangxi Province (20133BCB23020).
Appendix A. Supplementary Information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.09.001.
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