Biosensors and Bioelectronics 63 (2015) 425–431
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Ultrasensitive electrochemical detection of DNA based on Zn2 þ assistant DNA recycling followed with hybridization chain reaction dual amplification Yong Qian a, Chunyan Wang a, Fenglei Gao b,n a Key Laboratory of Radioactive Geology and Exploration Technology Fundamental Science for National Defense, East China Institute of Technology, Nanchang 344000, China b Jiangsu Key Laboratory of Target Drug and Clinical Application, School of Pharmacy, Xuzhou Medical College, Xuzhou 221004, China
art ic l e i nf o
a b s t r a c t
Article history: Received 21 April 2014 Received in revised form 29 July 2014 Accepted 30 July 2014 Available online 12 August 2014
A new strategy to combine Zn2 þ assistant DNA recycling followed with hybridization chain reaction dual amplification was designed for highly sensitive electrochemical detection of target DNA. A gold electrode was used to immobilize molecular beacon (MB) as the recognition probe and perform the amplification procedure. In the presence of the target DNA, the hairpin probe 1 was opened, and the DNAzyme was liberated from the caged structure. The activated DNAzyme hybridized with the MB and catalyzed its cleavage in the presence of Zn2 þ cofactor and resulting in a free DNAzyme strand. Finally, each targetinduced activated DNAzyme underwent many cycles triggering the cleavage of MB, thus forming numerous MB fragments. The MB fragments triggered the HCR and formed a long double-helix DNA structure. Because both H1 and H2 were labeled by biotin, a lot of SA-ALP was captured on the electrode surface, thus catalyzing a silver deposition process for electrochemical stripping analysis. This novel cascade signal amplification strategy can detect target DNA down to the attomolar level with a dynamic range spanning 6 orders of magnitude. This highly sensitive and specific assay has a great potential to become a promising DNA quantification method in biomedical research and clinical diagnosis. & 2014 Elsevier B.V. All rights reserved.
Keywords: Electrochemical Signal amplification Silver nanoparticles Hybridization chain reaction
1. Introduction Method for sensitive and selective detection of nucleic acids is of great importance in mutation identification, clinical diagnostics, and gene therapy (Hu et al., 2012; Huang et al., 2012; Yan et al., 2014). Various analytical approaches have been developed for DNA detection, including fluorescent (Dong et al., 2010; Zuo et al., 2010; Cheng et al., 2011; Wang et al., 2011a, 2011b; Ren et al., 2013), electrochemical (Xiao et al., 2009; Zhang et al., 2009; Hsieh et al., 2011; Zhu et al., 2013), colorimetric methods (Tang et al., 2012; Nie et al., 2014) and so on. Electrochemical techniques have gained great attention because of their relatively low cost, simplicity, sensitivity and ease of miniaturization. Electrochemical DNA assays commonly rely on the formation or disruption of the DNA double-helical structures. The electrochemical signals are produced at the electrode surface ascribing to the DNA probes modified with metal nanoparticles (Numnuam et al., 2008; Ting et al., 2009), enzymes (Hwang et al., 2005; Liu et al., 2008) or n
Corresponding author. Tel./fax: þ 86 516 83262138. E-mail address: jsxzgfl@sina.com (F. Gao).
http://dx.doi.org/10.1016/j.bios.2014.07.078 0956-5663/& 2014 Elsevier B.V. All rights reserved.
redox indicators (Liu et al., 2005; Jin et al., 2007) as the signal labels. Several signal amplification methods have been reported to achieve high sensitivity for DNA detection by loading a large number of signal labels on carriers such as graphene (Chen et al., 2012a, 2012b), carbon sphere (Dong et al., 2012) and carbon nanotubes (Wang et al., 2004; Kim et al., 2007; Huang et al., 2014), or by a catalytic redox-recycling (Limoges et al., 2008) amplification strategy. However, in above-mentioned assays for DNA detection, a single target molecule only reacts with a single signaling probe. This 1:1 hybridization ratio limits signal enhancement and its sensitivity. To overcome this problem, the strategies based on enzymeaided DNA amplification have been employed for DNA detection via different amplification approaches, such as target-induced displacement polymerization (Guo et al., 2009; Ren et al., 2010; Gao et al., 2013a, 2013b), ligase chain reaction (Shen et al., 2012), rolling circle amplification (Gao et al., 2013a, 2013b), polymerase chain reaction (Deng et al., 2012) and exonuclease (Zuo et al., 2010; Liu et al., 2014) or endonuclease (Xu et al., 2009; Chen et al., 2011) assisted DNA recycling that have realized the detection of target DNA at femtomolar levels. Moreover, the enzyme-free DNA
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amplification based on sequence-dependent DNA assembly has attracted much interest. For example, the hybridization chain reaction (HCR) (Chen et al., 2012a, 2012b), entropy-driven reaction (Zhang et al., 2007), and target-catalyzed hairpin assembly (Ju et al., 2012, Ma et al., 2012; Zheng et al., 2012) have been successfully used to achieve signal amplification. HCR driven by the free energy of base pair formation is a straightforward method for extending the DNA chain assemblies created by the triggered self-assembly of two stable hairpins species only in the presence of an initiator, which make HCR a fascinating strategy in signal amplification (Venkataraman et al., 2007). Recently, enzyme-free DNA amplification has also been accomplished by an autocatalytic and catabolic DNAzyme-mediated process (Wang et al., 2011a, 2011b). DNAzymes are nucleic acids isolated from combinatorial oligonucleotide libraries by in vitro selection. Similar to protein enzymes, DNAzymes show high catalytic hydrolytic cleavage activities towards specific substrates, while they are more stable than enzymes, and can be denatured and renatured many times without losing their catalytic activities toward substrates. This unique advantage makes DNAzymes ideal biocatalysts for amplified sensing applications. However, these amplification processes normally require complex signal probe conjugation or modification process (Lu et al., 2012). Thus the development of a simple method for amplified DNA is highly desired. In this study, we take advantage of the topological effect of DNAzyme, for the first time combining DNAzymes and HCR strategies to develop an electrochemical DNA sensor, in which DNA recycling and dual DNA amplification were successfully achieved. The proposed strategy exhibits high sensitivity and superior selectivity towards the target DNA, which may provide a universal sensing platform for DNA-based molecular diagnostics.
2. Experimental 2.1. Materials and reagents AgNO3 (99%) and mercaptoethanol (MCH, 98%) were purchased from Aldrich. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), cetyltrimethylammoniumbromide (CTAB), and p-Aminophenyl phosphate monohydrate (p-APP) were purchased from Sigma-Aldrich (St. Louis, MO). Streptavidin-alkaline phosphatase was obtained from Beyotime Biotechnology (Haimen, China). Human serum samples were kindly provided by the affiliated hospital of Xuzhou medical college (Xuzhou, China). A mixture containing equal volumes of human serum sample and DNA hybridization buffer was used for recovery testing. Ultrapure water obtained from a Millipore water purification system (18 MΩ, Milli-Q, Millipore) was used in all assays. Phosphate buffer saline (PBS) was prepared by mixing the stock solutions of NaH2PO4 and Na2HPO4 (10 mM, pH 7.4). DNA hybridization buffer was phosphate-buffered saline (137 mM NaCl, 2.5 mM Mg2 þ , 10 mM Na2HPO4, and 2.0 mM KH2PO4, pH 7.4). The binding buffer (BB) for associating with SA-ALP consisted of 50 mM Tris–HCl, 0.5 M NaCl, 0.1% Tween 20, and 1% bovine serum albumin (pH 7.4). The enzyme reaction buffer (EB) was 50 mM glycine, 1 mM MgSO4, and 1 mM ZnClO4 (pH 9.0). DNA oligonucleotides were synthesized and purified by Takara Biotechnology Co., Ltd. (Dalian, China) and stored in DNA hybridization buffer. The sequences of these oligonucleotides are as follows: Probe 1: 5′-CATCTCTTCTCCGAGCCGGTCGA AATAGTGGGTAATGAAGAGATGGTTGGTTGGT-3′ Target 1: 5′-CAACCAACCATCTCTTC-3′
Single-base mismatched: 5′-CAACCAACTATCTCTTC-3′ Two-base mismatched: 5′-CAGCCAACTATCTATTC-3′ Three-base mismatched: 5′-CAGCCAACTATCTATTC-3′ Non-complementary: 5′-TGCATCGGCAACCCAT-3′ MB: 5′-CCACCACATTGAAATTGACCCA CTATrAGGAAGAGATGTTACGAGGCGGTGGTGG-SH-3’ Hairpin probe 1 (H1): 5′-biotin-CGTAACATCTCTTC CCGTACTGGAAGAGAT biotin-3′ Hairpin probe 2 (H2): 3′-biotin-GCATGACCTTCTC TAGCATTGTAGAGAAGG -biotin-5′ Fluorophore-linker-Probe 1:
2.2. Immobilization of MB on the electrode surface The gold electrode (3 mm diameter) was firstly polished with 0.05 μm alumina powder to obtain a mirror surface, followed by sonication in water for 5 min. The gold electrode was then electrochemically cleaned in a 0.5 M H2SO4 solution with in a potential window between 0.2 and þ 1.5 V at a scan rate of 100 mV s 1 to remove any remaining impurities. The immobilization of MB on the electrode was performed in10 mM PBS solution containing 1 μM MB for 12 h at room temperature. Then, the electrode was immersed into 1 mM MCH for 1 h to remove the nonspecific DNA adsorption. The electrode surface was rinsed thoroughly and dried in nitrogen. 2.3. Dual amplification detection of target DNA To detect target DNA, the hybridization between probe 1 (5 μL, 0.1 μM) and different concentrations of target DNA (5 μL) was performed for 30 min. Then, 10 μL hybridization solutions and 2 μL of 0.8 mM Zn2 þ were dropped on the electrode surface for 80 min. Afterward, 10 μL of 10 mM Tris–HCl solution (pH 7.4, 500 mM NaCl, 1 mM MgCl2) containing 5 μM H1 and 5 μM H2 was dropped on the electrode surface for HCR, and incubated for 100 min, which was terminated through washing with PBS. The electrode was immersed in BB for 10 min to prevent the nonspecific adsorption of protein, and the resulting assembly was soaked in BB solution a containing 20 μg/mL SA-ALP for 30 min at room temperature, followed by two washes. Silver was deposited in EB solution containing 1.6 mM AgNO3 and 1.2 mM p-APP in a dark chamber for 25 min. After silver deposition, the electrode was rinsed with water and linear sweep stripping voltammetric (LSV) from 0.15 to 0.25 V at 50 mV s 1 was performed in a 1.0 M KCl solution. 2.4. Gel electrophoresis The 20% polyacrylamide gel electrophoresis (PAGE) analysis was carried out in 1 Tris–Borate–EDTA (pH 8.3) at a constant voltage of 100 V for about 2 h. After ethidium bromide staining, the gels were scanned using a Molecular Imager Gel Doc XR (BioRad). 2.5. Apparatus Electrochemical impedance spectroscopy (EIS) measurements were carried out on autolab PGSTAT12 (Ecochemie). All electrochemical experiments were performed with a conventional threeelectrode system comprising a gold working electrode, a platinum wire auxiliary electrode, and a Ag/AgCl reference electrode.
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Supporting electrolyte solution was 1.0 mmol/L K3[Fe(CN)6]/K4[Fe (CN)6] (1:1) solution containing 0.5 mol/L KCl. The ac voltage amplitude was 5 mV, and the voltage frequencies used for EIS measurements ranged from 10 kHz to 100 mHz. The applied potential was 172 mV vs. Ag/AgCl. The state of silver on the GE surface was characterized using scanning electron microscopy (SEM, FEI Sirion200).
3. Results and discussion 3.1. The principle of electrochemical sensor for amplification detection of DNA The principle of fabricated electrochemical DNA biosensor is shown in Scheme 1. First, a thiol-functionalized MB is immobilized onto the gold electrode surface by Au–S interaction. In the presence of the target DNA, the hairpin structure was opened, and the DNAzyme was liberated from the caged structure. The activated DNAzyme hybridized with the MB substrate and catalyzed in the presence of Zn2 þ cofactor (Brown et al., 2003). After the cleavage, the activated DNAzyme spontaneously dissociated from the surface of GE. Therefore, the amplification was accomplished by the hybridization of the activated DNAzyme with another MB to continue the strand-scission cycle, resulted in the cleavage of many MBs to form MB fragments. The sequence of the MB fragments triggered the HCR reaction and generated a cascade of hybridization to form a long double-helix DNA structure. Because both H1 and H2 probes are labeled by biotin, a lot of SA-ALP was capture on the electrode surface, thus converting the nonelectroactive substrate of the enzyme, p-APP, into the reducing agent, p-aminophenol. The latter, in turn, reduced silver ions leading to the deposition of the silver nanoparticles onto the electrode surface and DNA backbone for electrochemical stripping analysis. In the absence of target DNA, although probe 1 has some matched bases with the MB, they could not form a doublestranded structure because of the hairpin structure of probe 1. The novelty of the current study is the integration of these two emerging technologies for the fabrication of an ultrasensitive DNA biosensor. 3.2. Feasibility of electrochemical detection To estimate the signal amplification function of the proposed biosensor, the electrochemical performances of the sensor were investigated and compared under different conditions. As shown in Fig. 1A, when the GE sensors were incubated without target DNA before (blank, curve a) and after (curve b) HCR, only a little current responses were observed, on the GE. In the presence of target, and probe 1, the recognition of MB modified electrode to
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the activated DNAzyme after the HCR process, current did not increase (curve c) compared with blank, indicating that no HCR reaction was triggered. Subsequently, Zn2 þ was added to (b), the MB modified electrode hybridize with the activated DNAzyme after the HCR process led to an obvious stripping peak (curve d), which was greater than that in the absence of Zn2 þ , showing obvious signal amplification, which because the activated DNAzyme hybridized with the MB substrate and catalyzed the cleavage of the MB to form MB fragments in the presence of cofactor Zn2 þ for triggering the HCR. The HCR amplified the attachment of SAALP on the sensor surface and greatly improved the sensitivity of detection. Because the half-wave potential of p-AP is 0.097 V vs. NHE (Gyurcsanyi et al., 2002), and that of Ag þ is 0.80 V, p-AP spontaneously reduced silver ions in the solution. The reduction can be expressed as follows: 2Ag þ þp-AP-2Ag þQI, where QI (quinoimide) is the oxidation product of p-AP, caused by the loss of two electrons. In the silver deposition process, silver was deposited in EB solution containing 1.6 mM AgNO3 and 1.2 mM pAminophenyl phosphate monohydrate (p-APP), Av-ALP which binds to the exposed biotin group on the detection probes converts p-APP to a p-AP, a reducing agent that reduces silver ions for silver deposition. They do not produce silver deposition between Ag þ and p-APP, only the Av-ALP can start the reduction of silver ions in solution, so this method does not have large background. Moreover, although the oxidized form of p-AP is redox active in the potential window used in this study, paraaminophenol was rinsed out from the surface of GE by water after Ag deposition. The experimental results confirmed that Zn2 þ assisted DNA recycling and HCR occurred as expected for signal amplification. PAGE analysis was also used to investigate the viability of the sensing strategy (inset of Fig. 1A). Probe 1 exhibited only one band (lane a). After the target was added in the solution, a new band appeared (lane b), corresponding to the formed double stranded DNA. After MB was further added to the mixture, a new product band with a slow migration speed was observed (lane c). This result should be contributed to the product of MB–target–probe 1 DNA. After Zn2 þ was added, a new band appeared (lane d) with the fastest migration rate, which should be contributed to the small DNA pieces produced during the cleavage of MB by the Zn2 þ . After H1 and H2 were added, a new band with a slowest migration rate was observed in the end (lane e); this can be attributed to the obtained HCR product containing thousands of repeated sequences. On the other hand, the mixture of probe 1 and MB showed one band (lane f), indicating that probe 1 could not hybridize with the loop of the MB because the DNAzyme was caged by the hairpin structure of probe 1. The PAGE data demonstrated the feasibility of the designed strategy. To verify that the target DNA can open probe 1, hybridization tests were performed using free DNA strands in solution.
Scheme 1. Schematic illustration of the electrochemical DNA biosensor based on dual amplification of Zn2 þ assistant DNA recycling and hybridization chain reaction.
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Fig. 1. (A) LSV curves of Ag NPs deposited at sensor surface (50 mV s 1 in 1.0 M KCl) (a) blank, (b) 5 μM H1 and 5 μM H2, (c) 0.1 μM probe 1 and 0.1 pM target in the presence of H1 and H2, (d) 0.1 μM probe 1, 0.1 pM target, and 0.8 mM Zn2 þ in the presence of H1 and H2. Inset: PAGE analysis of the products with (a) 0.1 μM probe 1, (b) 0.1 μM probe 1, 0.1 μM target, (c) 0.1 μM probe 1, 0.1 μM target and 0.1 μM MB, (d) 0.1 μM probe 1, 0.1 μM target, 0.1 μM MB and 0.8 mM Zn2 þ , (e) 0.1 μM probe 1 and 0.1 μM MB; (f), (d) in the presence of H1 and H2. (B) Fluorescence spectra of (a) fluorophore-linker-probe 1, (b) fluorophore-linker-probe 1 and target DNA. Inset: Schematic representation of fluorescence verification strategy.
A fluorophore and a quencher were added to the probe 1. As shown in Fig. 1B, by itself or in the absence of target DNA, no fluorescence response was observed. However, upon the addition of target DNA, an enhanced fluorescence peak was observed, indicating that the target DNA could open the probe 1. Although the target only hybridized in the stem part of probe 1, the probe 1– target DNA duplex (19 base pairs) is more stable than the hairpin structure of probe 1 (9 base pairs); the target DNA will replace and free the stem of probe 1 when it hybridizes with probe 1. 3.3. Characterization of DNA sensors The DNA biosensor fabrication process was also confirmed by EIS measurements (Fig. 2). The typical electrochemical interface can be represented as an electrical circuit as shown in the inset of Fig. 2A. The components in the equivalent circuit included the solution resistance Rs, charge-transfer resistance Rct, constant phase element (CPE) related to double-layer capacitance, Warburg impedance Zw, resulting from the diffusion of ions from the bulk
electrolyte to the surface and Rw is the diffusion resistance. In the Nyquist plot of impedance spectroscopy, the diameter of the semicircle reveals the Rct. The fitting of the equivalent circuit to the experimental data yielded the solid lines drawn in Fig. 2A. The EIS of the bare electrode only exhibited a very small semicircular domain (curve a), indicating a fast charge-transfer process. After the capture probe was assembled onto the electrode surface, the semicircle increased remarkably, because the negatively charged phosphate backbone of assembled capture DNA prevented the diffusion of [Fe(CN)6]3 /4 towards the electrode surface (curve b). The Rct further increased after the target DNA activated DNAzyme was hybridized with MB, because an opened negatively charged interface electrostatically repelled the negatively charged [Fe(CN)6]3 /4 redox probe and inhibited interfacial charge transfer (curve c). After incubating with a mixture of the target, assistant probe and Zn2 þ , the resistance decreased dramatically (curve d). This resulted in not only the breakage of the capture probe but also the liberation of the assistant probe for recycling to cleave more of the capture probe, thus reducing the Rct.
Fig. 2. (A) EIS in 0.1 M KNO3 containing 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] at (a) bare GE, (b) immobilization of MB, (c) hybridization with target sequence, (d) after the hybridization of the probe 1 and target sequences, (d) after the hybridization of the probe 1 and target sequences in the presence of Zn2 þ for 80 min, and (e), (d) in the presence of H1 and H2 for 100 min. The inset shows the equivalent circuit applied to fit the impedance spectroscopy. (B) SEM image of the GE surface after silver deposition.
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Subsequently, a large semicircle domain was observed when the electrode was incubated with the H1 and H2 for HCR (curve e), indicating a high resistance of the electrode interface as the double helices copolymers that obstruct the electron transfer of the electrochemical probe.
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SEM was used to characterize the morphology of the silver deposition on GE surface. As shown in Fig. 2B, the GE surface produced abundant silver nanoparticles after the silver reduction. The sizes of the Ag nanoparticles are in the range of 60–90 nm, the shape of most Ag nanoparticles is sphere, and there is a good
Fig. 3. Dependence of current intensity for 0.1 pM target DNA on (A) the amount of p-APP, (B) the amount of Ag þ , (C) the amount of Zn2 þ , (D) the reaction time of Zn2 þ assistant DNA recycling, (E) the reaction time of HCR, and (F) the silver deposition time. When one parameter changes the others are under their optimal conditions.
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distribution as well as a high surface coverage onto GE because of protection by the high density DNA chain. 3.4. Optimization of detection conditions To achieve the best performance of the designed biosensor, the effects of the concentration of Zn2 þ , p-APP and Ag þ on the performance of the biosensor were investigated. The LSV response signals for target DNA (0.1 pM) are investigated with different pAPP, Ag þ , and Zn2 þ concentrations. As shown in Fig. 3A–C, the current responses increased with the increasing concentration and then levelled off, which corresponded to the saturated state. Consequently, the optimal amounts of p-APP, Ag þ , and Zn2 þ were selected at 1.2, 1.6 and 0.8 mM for the tests, respectively. To achieve the best performance of the designed biosensor, the reaction time of Zn2 þ assistant DNA recycling, HCR and the silver deposition time were also optimized. As shown in Fig. 3D, the peak current increased rapidly with the augment of the reaction duration because many MB substrates were cleavage. The maximum peak current was observed when the reaction was maintained at about 80 min. As shown in Fig. 3E, the peak current increased rapidly with the augment of the reaction duration because more repeated sequences could be formed and capture more of the SA-ALP. The maximum peak current was observed when the HCR reaction was maintained at about 100 min. As shown in Fig. 3F, the stripping current increased rapidly with the deposition time up to 25 min, indicating that 25 min was the optimal silver deposition time for the assay. 3.5. Analytical performance of the biosensor for target DNA Under the optimal conditions, the sensitivity of the sensor was performed by LSV measurements. Fig. 4A depicts LSV curves for varied concentrations of target DNA, which shows that the peak current increased with the increasing concentration of target DNA in the range from 10 aM to 10 pM. There was a good linear relationship between the peak current and the logarithm of the target DNA concentration in the range from 10 aM to 10 pM, as shown in Fig. 4B. The linear calibration equation was I¼ 315.9 þ 18.2 log C (I is the peak current (μA) and C is the concentration of the target DNA (mol L 1)) and the correlation coefficient R2 ¼0.9853. The detection limit was estimated to be 4.3 aM according to the 3s rule (where s is the standard deviation of the blank solutions, we use 3s as I, and put it into the formula (I ¼315.9 þ18.2 log C) for calculating the C value, the C was LOD).
The detection limit of this method was much lower than the combination of HCR with an electrochemical technique (0.5 fM) (Zhuang et al., 2013), Zn2 þ assistant DNA recycling with fluorescent spectroscopy (10 fM) (Wang et al., 2011a, 2011b), and competitive with the PCR technique (Deng et al., 2012). Thus the high sensitivity of this method could be attributed to three factors: (1) Zn2 þ -assisted DNA recycling for formation of many MB fragments to trigger HCR, (2) highly loaded enzyme molecules on the surface of electrode by HCR, and (3) enzymatic reaction for silver deposition, and the stripping analysis of deposited silver. Thus the results identified that this signal amplification method was efficient for ultrasensitive electrochemical detection of DNA hybridization. In order to evaluate the selectivity of this biosensor, five different DNA sequences (1.0 fM) were investigated including single-base mismatched, two-base mismatcht, three-base mismatched, noncomplementary mismatched target and blank. The perfect complementary target showed a signal of 13.5-fold, 17.1fold, 18.9-fold and 23.4 of single-base, two-base, and three-base mismatched and noncomplementary oligonucleotide, respectively (Fig. 5). These results indicate that the employment of four hairpin probes, MB, probe 1, H1 and H2 for the proposed amplified biosensor significantly improved the ability of single nucleotide polymorphisms identification. 3.6. Reproducibility for target DNA detection The reproducibility of the suggested electrochemical detection method was examined by six repetitive measurements of 0.1 pM target DNA on a single electrode, which showed a relative standard deviation (RSD) of 2.6%. The RSD for six parallel DNA sensors fabricated on different electrodes was 5.7%. These results indicated the satisfactory reproducibility for both DNA detection and DNA sensor fabrication. 3.7. Application in real sample To test the generality of the proposed assay in the clinical sample, we imitate the same environment with real sample by spiking target DNA solution into human serum due to lack of the real sample (Huang et al., 2014; Zhou et al., 2014). The highly concentrated tissue in human serum would affect the hybridization efficiency of DNA, so we use a diluted human serum (10%) during real sample testing. At the concentrations of 0.1 pM and 0.1 fM, the recoveries were 96.3 71.7% and 94.17 3.4% (n ¼3),
Fig. 4. (A) LSV curves of the DNA sensors toward target DNA with various levels in 1.0 M KCl. (B) The corresponding calibration curve.
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Fig. 5. Histograms of current intensity for 1.0 fM (a) complementary, (b) singlebase mismatched, (c) two-base mismatched, (d) three-base mismatched, (e) fourbase mismatched sequences, and (f) non-complementary.
indicating that the proposed strategy for DNA detection could be applied in real sample analysis.
4. Conclusion This work has proposed a novel, and isothermal electrochemical platform for ultrasensitive detection of target DNA based on the combination of Zn2 þ assistant DNA recycling and HCR strategies. Zn2 þ assistant DNA recycling can produce a large number of cleaved MB fragments with the initiation of target DNA, which then initiated the reaction of the HCR for realizing the dual signal amplification. The amount of target DNA can be easily read out through the electrochemical stripping analysis silver with LSV measurements. By integrating molecular biological technology, nanobiotechnology, and electrochemical detection, this novel cascade signal amplification strategy can detect target DNA down to the attomolar level with high selectivity to differentiate mismatched DNA. In view of the advantages, the proposed strategy can be extended to a versatile amplified sensing platform for molecular diagnostics in complex biological systems.
Acknowledgment This work was supported by the National Natural Science Foundation of China (Nos. 21264001 and 21004009), Excellent Talents of Xuzhou Medical College (D2014007), Natural Science Foundation of Jiangxi Province (20114BAB213010) and Young Scientist Foundation of Jiangxi Province (20133BCB23020).
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Ultrasensitive electrochemical detection of DNA based on Zn2 þ assistant DNA recycling followed with hybridization chain reaction dual amplification Yong Qian a, Chunyan Wang a, Fenglei Gao b,n a Key Laboratory of Radioactive Geology and Exploration Technology Fundamental Science for National Defense, East China Institute of Technology, Nanchang 344000, China b Jiangsu Key Laboratory of Target Drug and Clinical Application, School of Pharmacy, Xuzhou Medical College, Xuzhou 221004, China
art ic l e i nf o
a b s t r a c t
Article history: Received 21 April 2014 Received in revised form 29 July 2014 Accepted 30 July 2014 Available online 12 August 2014
A new strategy to combine Zn2 þ assistant DNA recycling followed with hybridization chain reaction dual amplification was designed for highly sensitive electrochemical detection of target DNA. A gold electrode was used to immobilize molecular beacon (MB) as the recognition probe and perform the amplification procedure. In the presence of the target DNA, the hairpin probe 1 was opened, and the DNAzyme was liberated from the caged structure. The activated DNAzyme hybridized with the MB and catalyzed its cleavage in the presence of Zn2 þ cofactor and resulting in a free DNAzyme strand. Finally, each targetinduced activated DNAzyme underwent many cycles triggering the cleavage of MB, thus forming numerous MB fragments. The MB fragments triggered the HCR and formed a long double-helix DNA structure. Because both H1 and H2 were labeled by biotin, a lot of SA-ALP was captured on the electrode surface, thus catalyzing a silver deposition process for electrochemical stripping analysis. This novel cascade signal amplification strategy can detect target DNA down to the attomolar level with a dynamic range spanning 6 orders of magnitude. This highly sensitive and specific assay has a great potential to become a promising DNA quantification method in biomedical research and clinical diagnosis. & 2014 Elsevier B.V. All rights reserved.
Keywords: Electrochemical Signal amplification Silver nanoparticles Hybridization chain reaction
1. Introduction Method for sensitive and selective detection of nucleic acids is of great importance in mutation identification, clinical diagnostics, and gene therapy (Hu et al., 2012; Huang et al., 2012; Yan et al., 2014). Various analytical approaches have been developed for DNA detection, including fluorescent (Dong et al., 2010; Zuo et al., 2010; Cheng et al., 2011; Wang et al., 2011a, 2011b; Ren et al., 2013), electrochemical (Xiao et al., 2009; Zhang et al., 2009; Hsieh et al., 2011; Zhu et al., 2013), colorimetric methods (Tang et al., 2012; Nie et al., 2014) and so on. Electrochemical techniques have gained great attention because of their relatively low cost, simplicity, sensitivity and ease of miniaturization. Electrochemical DNA assays commonly rely on the formation or disruption of the DNA double-helical structures. The electrochemical signals are produced at the electrode surface ascribing to the DNA probes modified with metal nanoparticles (Numnuam et al., 2008; Ting et al., 2009), enzymes (Hwang et al., 2005; Liu et al., 2008) or n
Corresponding author. Tel./fax: þ 86 516 83262138. E-mail address: jsxzgfl@sina.com (F. Gao).
http://dx.doi.org/10.1016/j.bios.2014.07.078 0956-5663/& 2014 Elsevier B.V. All rights reserved.
redox indicators (Liu et al., 2005; Jin et al., 2007) as the signal labels. Several signal amplification methods have been reported to achieve high sensitivity for DNA detection by loading a large number of signal labels on carriers such as graphene (Chen et al., 2012a, 2012b), carbon sphere (Dong et al., 2012) and carbon nanotubes (Wang et al., 2004; Kim et al., 2007; Huang et al., 2014), or by a catalytic redox-recycling (Limoges et al., 2008) amplification strategy. However, in above-mentioned assays for DNA detection, a single target molecule only reacts with a single signaling probe. This 1:1 hybridization ratio limits signal enhancement and its sensitivity. To overcome this problem, the strategies based on enzymeaided DNA amplification have been employed for DNA detection via different amplification approaches, such as target-induced displacement polymerization (Guo et al., 2009; Ren et al., 2010; Gao et al., 2013a, 2013b), ligase chain reaction (Shen et al., 2012), rolling circle amplification (Gao et al., 2013a, 2013b), polymerase chain reaction (Deng et al., 2012) and exonuclease (Zuo et al., 2010; Liu et al., 2014) or endonuclease (Xu et al., 2009; Chen et al., 2011) assisted DNA recycling that have realized the detection of target DNA at femtomolar levels. Moreover, the enzyme-free DNA
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amplification based on sequence-dependent DNA assembly has attracted much interest. For example, the hybridization chain reaction (HCR) (Chen et al., 2012a, 2012b), entropy-driven reaction (Zhang et al., 2007), and target-catalyzed hairpin assembly (Ju et al., 2012, Ma et al., 2012; Zheng et al., 2012) have been successfully used to achieve signal amplification. HCR driven by the free energy of base pair formation is a straightforward method for extending the DNA chain assemblies created by the triggered self-assembly of two stable hairpins species only in the presence of an initiator, which make HCR a fascinating strategy in signal amplification (Venkataraman et al., 2007). Recently, enzyme-free DNA amplification has also been accomplished by an autocatalytic and catabolic DNAzyme-mediated process (Wang et al., 2011a, 2011b). DNAzymes are nucleic acids isolated from combinatorial oligonucleotide libraries by in vitro selection. Similar to protein enzymes, DNAzymes show high catalytic hydrolytic cleavage activities towards specific substrates, while they are more stable than enzymes, and can be denatured and renatured many times without losing their catalytic activities toward substrates. This unique advantage makes DNAzymes ideal biocatalysts for amplified sensing applications. However, these amplification processes normally require complex signal probe conjugation or modification process (Lu et al., 2012). Thus the development of a simple method for amplified DNA is highly desired. In this study, we take advantage of the topological effect of DNAzyme, for the first time combining DNAzymes and HCR strategies to develop an electrochemical DNA sensor, in which DNA recycling and dual DNA amplification were successfully achieved. The proposed strategy exhibits high sensitivity and superior selectivity towards the target DNA, which may provide a universal sensing platform for DNA-based molecular diagnostics.
2. Experimental 2.1. Materials and reagents AgNO3 (99%) and mercaptoethanol (MCH, 98%) were purchased from Aldrich. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), cetyltrimethylammoniumbromide (CTAB), and p-Aminophenyl phosphate monohydrate (p-APP) were purchased from Sigma-Aldrich (St. Louis, MO). Streptavidin-alkaline phosphatase was obtained from Beyotime Biotechnology (Haimen, China). Human serum samples were kindly provided by the affiliated hospital of Xuzhou medical college (Xuzhou, China). A mixture containing equal volumes of human serum sample and DNA hybridization buffer was used for recovery testing. Ultrapure water obtained from a Millipore water purification system (18 MΩ, Milli-Q, Millipore) was used in all assays. Phosphate buffer saline (PBS) was prepared by mixing the stock solutions of NaH2PO4 and Na2HPO4 (10 mM, pH 7.4). DNA hybridization buffer was phosphate-buffered saline (137 mM NaCl, 2.5 mM Mg2 þ , 10 mM Na2HPO4, and 2.0 mM KH2PO4, pH 7.4). The binding buffer (BB) for associating with SA-ALP consisted of 50 mM Tris–HCl, 0.5 M NaCl, 0.1% Tween 20, and 1% bovine serum albumin (pH 7.4). The enzyme reaction buffer (EB) was 50 mM glycine, 1 mM MgSO4, and 1 mM ZnClO4 (pH 9.0). DNA oligonucleotides were synthesized and purified by Takara Biotechnology Co., Ltd. (Dalian, China) and stored in DNA hybridization buffer. The sequences of these oligonucleotides are as follows: Probe 1: 5′-CATCTCTTCTCCGAGCCGGTCGA AATAGTGGGTAATGAAGAGATGGTTGGTTGGT-3′ Target 1: 5′-CAACCAACCATCTCTTC-3′
Single-base mismatched: 5′-CAACCAACTATCTCTTC-3′ Two-base mismatched: 5′-CAGCCAACTATCTATTC-3′ Three-base mismatched: 5′-CAGCCAACTATCTATTC-3′ Non-complementary: 5′-TGCATCGGCAACCCAT-3′ MB: 5′-CCACCACATTGAAATTGACCCA CTATrAGGAAGAGATGTTACGAGGCGGTGGTGG-SH-3’ Hairpin probe 1 (H1): 5′-biotin-CGTAACATCTCTTC CCGTACTGGAAGAGAT biotin-3′ Hairpin probe 2 (H2): 3′-biotin-GCATGACCTTCTC TAGCATTGTAGAGAAGG -biotin-5′ Fluorophore-linker-Probe 1:
2.2. Immobilization of MB on the electrode surface The gold electrode (3 mm diameter) was firstly polished with 0.05 μm alumina powder to obtain a mirror surface, followed by sonication in water for 5 min. The gold electrode was then electrochemically cleaned in a 0.5 M H2SO4 solution with in a potential window between 0.2 and þ 1.5 V at a scan rate of 100 mV s 1 to remove any remaining impurities. The immobilization of MB on the electrode was performed in10 mM PBS solution containing 1 μM MB for 12 h at room temperature. Then, the electrode was immersed into 1 mM MCH for 1 h to remove the nonspecific DNA adsorption. The electrode surface was rinsed thoroughly and dried in nitrogen. 2.3. Dual amplification detection of target DNA To detect target DNA, the hybridization between probe 1 (5 μL, 0.1 μM) and different concentrations of target DNA (5 μL) was performed for 30 min. Then, 10 μL hybridization solutions and 2 μL of 0.8 mM Zn2 þ were dropped on the electrode surface for 80 min. Afterward, 10 μL of 10 mM Tris–HCl solution (pH 7.4, 500 mM NaCl, 1 mM MgCl2) containing 5 μM H1 and 5 μM H2 was dropped on the electrode surface for HCR, and incubated for 100 min, which was terminated through washing with PBS. The electrode was immersed in BB for 10 min to prevent the nonspecific adsorption of protein, and the resulting assembly was soaked in BB solution a containing 20 μg/mL SA-ALP for 30 min at room temperature, followed by two washes. Silver was deposited in EB solution containing 1.6 mM AgNO3 and 1.2 mM p-APP in a dark chamber for 25 min. After silver deposition, the electrode was rinsed with water and linear sweep stripping voltammetric (LSV) from 0.15 to 0.25 V at 50 mV s 1 was performed in a 1.0 M KCl solution. 2.4. Gel electrophoresis The 20% polyacrylamide gel electrophoresis (PAGE) analysis was carried out in 1 Tris–Borate–EDTA (pH 8.3) at a constant voltage of 100 V for about 2 h. After ethidium bromide staining, the gels were scanned using a Molecular Imager Gel Doc XR (BioRad). 2.5. Apparatus Electrochemical impedance spectroscopy (EIS) measurements were carried out on autolab PGSTAT12 (Ecochemie). All electrochemical experiments were performed with a conventional threeelectrode system comprising a gold working electrode, a platinum wire auxiliary electrode, and a Ag/AgCl reference electrode.
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Supporting electrolyte solution was 1.0 mmol/L K3[Fe(CN)6]/K4[Fe (CN)6] (1:1) solution containing 0.5 mol/L KCl. The ac voltage amplitude was 5 mV, and the voltage frequencies used for EIS measurements ranged from 10 kHz to 100 mHz. The applied potential was 172 mV vs. Ag/AgCl. The state of silver on the GE surface was characterized using scanning electron microscopy (SEM, FEI Sirion200).
3. Results and discussion 3.1. The principle of electrochemical sensor for amplification detection of DNA The principle of fabricated electrochemical DNA biosensor is shown in Scheme 1. First, a thiol-functionalized MB is immobilized onto the gold electrode surface by Au–S interaction. In the presence of the target DNA, the hairpin structure was opened, and the DNAzyme was liberated from the caged structure. The activated DNAzyme hybridized with the MB substrate and catalyzed in the presence of Zn2 þ cofactor (Brown et al., 2003). After the cleavage, the activated DNAzyme spontaneously dissociated from the surface of GE. Therefore, the amplification was accomplished by the hybridization of the activated DNAzyme with another MB to continue the strand-scission cycle, resulted in the cleavage of many MBs to form MB fragments. The sequence of the MB fragments triggered the HCR reaction and generated a cascade of hybridization to form a long double-helix DNA structure. Because both H1 and H2 probes are labeled by biotin, a lot of SA-ALP was capture on the electrode surface, thus converting the nonelectroactive substrate of the enzyme, p-APP, into the reducing agent, p-aminophenol. The latter, in turn, reduced silver ions leading to the deposition of the silver nanoparticles onto the electrode surface and DNA backbone for electrochemical stripping analysis. In the absence of target DNA, although probe 1 has some matched bases with the MB, they could not form a doublestranded structure because of the hairpin structure of probe 1. The novelty of the current study is the integration of these two emerging technologies for the fabrication of an ultrasensitive DNA biosensor. 3.2. Feasibility of electrochemical detection To estimate the signal amplification function of the proposed biosensor, the electrochemical performances of the sensor were investigated and compared under different conditions. As shown in Fig. 1A, when the GE sensors were incubated without target DNA before (blank, curve a) and after (curve b) HCR, only a little current responses were observed, on the GE. In the presence of target, and probe 1, the recognition of MB modified electrode to
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the activated DNAzyme after the HCR process, current did not increase (curve c) compared with blank, indicating that no HCR reaction was triggered. Subsequently, Zn2 þ was added to (b), the MB modified electrode hybridize with the activated DNAzyme after the HCR process led to an obvious stripping peak (curve d), which was greater than that in the absence of Zn2 þ , showing obvious signal amplification, which because the activated DNAzyme hybridized with the MB substrate and catalyzed the cleavage of the MB to form MB fragments in the presence of cofactor Zn2 þ for triggering the HCR. The HCR amplified the attachment of SAALP on the sensor surface and greatly improved the sensitivity of detection. Because the half-wave potential of p-AP is 0.097 V vs. NHE (Gyurcsanyi et al., 2002), and that of Ag þ is 0.80 V, p-AP spontaneously reduced silver ions in the solution. The reduction can be expressed as follows: 2Ag þ þp-AP-2Ag þQI, where QI (quinoimide) is the oxidation product of p-AP, caused by the loss of two electrons. In the silver deposition process, silver was deposited in EB solution containing 1.6 mM AgNO3 and 1.2 mM pAminophenyl phosphate monohydrate (p-APP), Av-ALP which binds to the exposed biotin group on the detection probes converts p-APP to a p-AP, a reducing agent that reduces silver ions for silver deposition. They do not produce silver deposition between Ag þ and p-APP, only the Av-ALP can start the reduction of silver ions in solution, so this method does not have large background. Moreover, although the oxidized form of p-AP is redox active in the potential window used in this study, paraaminophenol was rinsed out from the surface of GE by water after Ag deposition. The experimental results confirmed that Zn2 þ assisted DNA recycling and HCR occurred as expected for signal amplification. PAGE analysis was also used to investigate the viability of the sensing strategy (inset of Fig. 1A). Probe 1 exhibited only one band (lane a). After the target was added in the solution, a new band appeared (lane b), corresponding to the formed double stranded DNA. After MB was further added to the mixture, a new product band with a slow migration speed was observed (lane c). This result should be contributed to the product of MB–target–probe 1 DNA. After Zn2 þ was added, a new band appeared (lane d) with the fastest migration rate, which should be contributed to the small DNA pieces produced during the cleavage of MB by the Zn2 þ . After H1 and H2 were added, a new band with a slowest migration rate was observed in the end (lane e); this can be attributed to the obtained HCR product containing thousands of repeated sequences. On the other hand, the mixture of probe 1 and MB showed one band (lane f), indicating that probe 1 could not hybridize with the loop of the MB because the DNAzyme was caged by the hairpin structure of probe 1. The PAGE data demonstrated the feasibility of the designed strategy. To verify that the target DNA can open probe 1, hybridization tests were performed using free DNA strands in solution.
Scheme 1. Schematic illustration of the electrochemical DNA biosensor based on dual amplification of Zn2 þ assistant DNA recycling and hybridization chain reaction.
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Fig. 1. (A) LSV curves of Ag NPs deposited at sensor surface (50 mV s 1 in 1.0 M KCl) (a) blank, (b) 5 μM H1 and 5 μM H2, (c) 0.1 μM probe 1 and 0.1 pM target in the presence of H1 and H2, (d) 0.1 μM probe 1, 0.1 pM target, and 0.8 mM Zn2 þ in the presence of H1 and H2. Inset: PAGE analysis of the products with (a) 0.1 μM probe 1, (b) 0.1 μM probe 1, 0.1 μM target, (c) 0.1 μM probe 1, 0.1 μM target and 0.1 μM MB, (d) 0.1 μM probe 1, 0.1 μM target, 0.1 μM MB and 0.8 mM Zn2 þ , (e) 0.1 μM probe 1 and 0.1 μM MB; (f), (d) in the presence of H1 and H2. (B) Fluorescence spectra of (a) fluorophore-linker-probe 1, (b) fluorophore-linker-probe 1 and target DNA. Inset: Schematic representation of fluorescence verification strategy.
A fluorophore and a quencher were added to the probe 1. As shown in Fig. 1B, by itself or in the absence of target DNA, no fluorescence response was observed. However, upon the addition of target DNA, an enhanced fluorescence peak was observed, indicating that the target DNA could open the probe 1. Although the target only hybridized in the stem part of probe 1, the probe 1– target DNA duplex (19 base pairs) is more stable than the hairpin structure of probe 1 (9 base pairs); the target DNA will replace and free the stem of probe 1 when it hybridizes with probe 1. 3.3. Characterization of DNA sensors The DNA biosensor fabrication process was also confirmed by EIS measurements (Fig. 2). The typical electrochemical interface can be represented as an electrical circuit as shown in the inset of Fig. 2A. The components in the equivalent circuit included the solution resistance Rs, charge-transfer resistance Rct, constant phase element (CPE) related to double-layer capacitance, Warburg impedance Zw, resulting from the diffusion of ions from the bulk
electrolyte to the surface and Rw is the diffusion resistance. In the Nyquist plot of impedance spectroscopy, the diameter of the semicircle reveals the Rct. The fitting of the equivalent circuit to the experimental data yielded the solid lines drawn in Fig. 2A. The EIS of the bare electrode only exhibited a very small semicircular domain (curve a), indicating a fast charge-transfer process. After the capture probe was assembled onto the electrode surface, the semicircle increased remarkably, because the negatively charged phosphate backbone of assembled capture DNA prevented the diffusion of [Fe(CN)6]3 /4 towards the electrode surface (curve b). The Rct further increased after the target DNA activated DNAzyme was hybridized with MB, because an opened negatively charged interface electrostatically repelled the negatively charged [Fe(CN)6]3 /4 redox probe and inhibited interfacial charge transfer (curve c). After incubating with a mixture of the target, assistant probe and Zn2 þ , the resistance decreased dramatically (curve d). This resulted in not only the breakage of the capture probe but also the liberation of the assistant probe for recycling to cleave more of the capture probe, thus reducing the Rct.
Fig. 2. (A) EIS in 0.1 M KNO3 containing 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] at (a) bare GE, (b) immobilization of MB, (c) hybridization with target sequence, (d) after the hybridization of the probe 1 and target sequences, (d) after the hybridization of the probe 1 and target sequences in the presence of Zn2 þ for 80 min, and (e), (d) in the presence of H1 and H2 for 100 min. The inset shows the equivalent circuit applied to fit the impedance spectroscopy. (B) SEM image of the GE surface after silver deposition.
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Subsequently, a large semicircle domain was observed when the electrode was incubated with the H1 and H2 for HCR (curve e), indicating a high resistance of the electrode interface as the double helices copolymers that obstruct the electron transfer of the electrochemical probe.
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SEM was used to characterize the morphology of the silver deposition on GE surface. As shown in Fig. 2B, the GE surface produced abundant silver nanoparticles after the silver reduction. The sizes of the Ag nanoparticles are in the range of 60–90 nm, the shape of most Ag nanoparticles is sphere, and there is a good
Fig. 3. Dependence of current intensity for 0.1 pM target DNA on (A) the amount of p-APP, (B) the amount of Ag þ , (C) the amount of Zn2 þ , (D) the reaction time of Zn2 þ assistant DNA recycling, (E) the reaction time of HCR, and (F) the silver deposition time. When one parameter changes the others are under their optimal conditions.
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distribution as well as a high surface coverage onto GE because of protection by the high density DNA chain. 3.4. Optimization of detection conditions To achieve the best performance of the designed biosensor, the effects of the concentration of Zn2 þ , p-APP and Ag þ on the performance of the biosensor were investigated. The LSV response signals for target DNA (0.1 pM) are investigated with different pAPP, Ag þ , and Zn2 þ concentrations. As shown in Fig. 3A–C, the current responses increased with the increasing concentration and then levelled off, which corresponded to the saturated state. Consequently, the optimal amounts of p-APP, Ag þ , and Zn2 þ were selected at 1.2, 1.6 and 0.8 mM for the tests, respectively. To achieve the best performance of the designed biosensor, the reaction time of Zn2 þ assistant DNA recycling, HCR and the silver deposition time were also optimized. As shown in Fig. 3D, the peak current increased rapidly with the augment of the reaction duration because many MB substrates were cleavage. The maximum peak current was observed when the reaction was maintained at about 80 min. As shown in Fig. 3E, the peak current increased rapidly with the augment of the reaction duration because more repeated sequences could be formed and capture more of the SA-ALP. The maximum peak current was observed when the HCR reaction was maintained at about 100 min. As shown in Fig. 3F, the stripping current increased rapidly with the deposition time up to 25 min, indicating that 25 min was the optimal silver deposition time for the assay. 3.5. Analytical performance of the biosensor for target DNA Under the optimal conditions, the sensitivity of the sensor was performed by LSV measurements. Fig. 4A depicts LSV curves for varied concentrations of target DNA, which shows that the peak current increased with the increasing concentration of target DNA in the range from 10 aM to 10 pM. There was a good linear relationship between the peak current and the logarithm of the target DNA concentration in the range from 10 aM to 10 pM, as shown in Fig. 4B. The linear calibration equation was I¼ 315.9 þ 18.2 log C (I is the peak current (μA) and C is the concentration of the target DNA (mol L 1)) and the correlation coefficient R2 ¼0.9853. The detection limit was estimated to be 4.3 aM according to the 3s rule (where s is the standard deviation of the blank solutions, we use 3s as I, and put it into the formula (I ¼315.9 þ18.2 log C) for calculating the C value, the C was LOD).
The detection limit of this method was much lower than the combination of HCR with an electrochemical technique (0.5 fM) (Zhuang et al., 2013), Zn2 þ assistant DNA recycling with fluorescent spectroscopy (10 fM) (Wang et al., 2011a, 2011b), and competitive with the PCR technique (Deng et al., 2012). Thus the high sensitivity of this method could be attributed to three factors: (1) Zn2 þ -assisted DNA recycling for formation of many MB fragments to trigger HCR, (2) highly loaded enzyme molecules on the surface of electrode by HCR, and (3) enzymatic reaction for silver deposition, and the stripping analysis of deposited silver. Thus the results identified that this signal amplification method was efficient for ultrasensitive electrochemical detection of DNA hybridization. In order to evaluate the selectivity of this biosensor, five different DNA sequences (1.0 fM) were investigated including single-base mismatched, two-base mismatcht, three-base mismatched, noncomplementary mismatched target and blank. The perfect complementary target showed a signal of 13.5-fold, 17.1fold, 18.9-fold and 23.4 of single-base, two-base, and three-base mismatched and noncomplementary oligonucleotide, respectively (Fig. 5). These results indicate that the employment of four hairpin probes, MB, probe 1, H1 and H2 for the proposed amplified biosensor significantly improved the ability of single nucleotide polymorphisms identification. 3.6. Reproducibility for target DNA detection The reproducibility of the suggested electrochemical detection method was examined by six repetitive measurements of 0.1 pM target DNA on a single electrode, which showed a relative standard deviation (RSD) of 2.6%. The RSD for six parallel DNA sensors fabricated on different electrodes was 5.7%. These results indicated the satisfactory reproducibility for both DNA detection and DNA sensor fabrication. 3.7. Application in real sample To test the generality of the proposed assay in the clinical sample, we imitate the same environment with real sample by spiking target DNA solution into human serum due to lack of the real sample (Huang et al., 2014; Zhou et al., 2014). The highly concentrated tissue in human serum would affect the hybridization efficiency of DNA, so we use a diluted human serum (10%) during real sample testing. At the concentrations of 0.1 pM and 0.1 fM, the recoveries were 96.3 71.7% and 94.17 3.4% (n ¼3),
Fig. 4. (A) LSV curves of the DNA sensors toward target DNA with various levels in 1.0 M KCl. (B) The corresponding calibration curve.
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Fig. 5. Histograms of current intensity for 1.0 fM (a) complementary, (b) singlebase mismatched, (c) two-base mismatched, (d) three-base mismatched, (e) fourbase mismatched sequences, and (f) non-complementary.
indicating that the proposed strategy for DNA detection could be applied in real sample analysis.
4. Conclusion This work has proposed a novel, and isothermal electrochemical platform for ultrasensitive detection of target DNA based on the combination of Zn2 þ assistant DNA recycling and HCR strategies. Zn2 þ assistant DNA recycling can produce a large number of cleaved MB fragments with the initiation of target DNA, which then initiated the reaction of the HCR for realizing the dual signal amplification. The amount of target DNA can be easily read out through the electrochemical stripping analysis silver with LSV measurements. By integrating molecular biological technology, nanobiotechnology, and electrochemical detection, this novel cascade signal amplification strategy can detect target DNA down to the attomolar level with high selectivity to differentiate mismatched DNA. In view of the advantages, the proposed strategy can be extended to a versatile amplified sensing platform for molecular diagnostics in complex biological systems.
Acknowledgment This work was supported by the National Natural Science Foundation of China (Nos. 21264001 and 21004009), Excellent Talents of Xuzhou Medical College (D2014007), Natural Science Foundation of Jiangxi Province (20114BAB213010) and Young Scientist Foundation of Jiangxi Province (20133BCB23020).
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