Analytica Chimica Acta 862 (2015) 64–69
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
A sensitive electrochemical aptasensor for ATP detection based on exonuclease III-assisted signal amplification strategy Ting Bao 1, Huawei Shu 1, Wei Wen, Xiuhua Zhang, Shengfu Wang * Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Ministry of Education Key Laboratory for the Synthesis and Application of Organic Functional Molecules & College of Chemistry and Chemical Engineering, Hubei University, Wuhan 430062, PR China
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
Distinctive sequence of hairpinaptamer was designed. Structure-switching of aptamer to reveal the MB labeled terminus. Exonuclease-catalyzed target recycling amplified signal significantly. Exo III improved the sensitivity of the aptasensor significantly.
Illustration of the stepwise aptasensor fabrication based on exonuclease-assisted target recycling for amplification.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 November 2014 Received in revised form 26 December 2014 Accepted 28 December 2014 Available online 30 December 2014
A target-induced structure-switching electrochemical aptasensor for sensitive detection of ATP was successfully constructed which was based on exonuclease III-catalyzed target recycling for signal amplification. With the existence of ATP, methylene blue (MB) labeled hairpin DNA formed G-quadruplex with ATP, which led to conformational changes of the hairpin DNA and created catalytic cleavage sites for exonuclease III (Exo III). Then the structure-switching DNA hybridized with capture DNA which made MB close to electrode surface. Meanwhile, Exo III selectively digested aptamer from its 30 -end, thus G-quadruplex structure was destroyed and ATP was released for target recycling. The Exo III-assisted target recycling amplified electrochemical signal significantly. Fluorescence experiment was performed to confirm the structure-switching process of the hairpin DNA. In fluorescence experiment, AuNPs–aptamer conjugates were synthesized, AuNPs quenched fluorescence of MB, the target-induced structure-switching made Exo III digested aptamer, which restored fluorescence. Under optimized conditions, the proposed aptasensor showed a linear range of 0.1–20 nM with a detection limit of 34 pM. In addition, the proposed aptasensor had good stability and selectivity, offered promising choice for the detection of other small molecules. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Aptamer Electrochemical aptasensor Exonuclease III Signal amplification Adenosine triphosphate
1. Introduction Adenosine triphosphate (ATP) is a multifunctional nucleoside which works as cellular energy currency in living cells [1]. It is
* Corresponding author. Tel.: +86 27 50865309; fax: +86 27 88663043. E-mail address: [email protected] (S. Wang). Both authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.aca.2014.12.049 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.
used as an indicator for cell viability and cell injury and plays a significant role in metabolism and many enzymatic reactions, such as cellular respiration and ions transportation in cells [2,3]. The concentration change of ATP in cells was in connection with many diseases, for instance, angiocardiopathy and Parkinson’s diseases [4,5]. Therefore, sensitive detection of ATP is of great importance in biochemical analysis and clinical diagnoses. Different methods have been used for the detection of ATP, such as ultra performance liquid chromatography [6], fluorescence [7],
T. Bao et al. / Analytica Chimica Acta 862 (2015) 64–69
electrochemiluminescent (ECL) [8] and UV–vis reflectance spectrum [9]. However, limited by their high cost, complex and time-consuming testing process, these methods are not satisfactory. Nevertheless, electrochemical aptasensors have the advantage of low-cost and fast-response, which are proved to be an excellent tool for ATP detection [10]. Aptamers are synthetic single-stranded DNA or RNA which are artificially selected by systematic evolution of ligands by exponential enrichment (SELEX), which could specially recognize diverse targets including protein, metal ions, small molecules and cells [11,12]. ATP aptamer can recognize and bind with ATP to form G-quadruplex structure, it has high affinity and selectivity to ATP [13]. Therefore ATP aptamer is widely used to construct electrochemical aptasensor for sensitive detection of ATP [14,15]. Gold nanoparticles, graphene and exonuclease have been used to amplify the signal of aptasensor. Among them, exonucleasecatalyzed target recycling has been proved to be a perfect method for signal amplification [16–18]. Exonuclease III (Exo III) can selectively digest double-stranded DNA from its 30 -end, which led to the destruction of the aptamer structure and the analyte was released into solution to participate new recycling process [19–21]. In this way, the signal was amplified significantly. Herein, a hairpin DNA consisted of an aptamer sequence for ATP and methylene blue (MB) labeled at the 50 -terminus was ingeniously designed. Exo III could not cleave the hairpin DNA because the 30 -end of hairpin DNA was single-stranded. With the addition of ATP, the hairpin DNA reconfigured to G-quadruplex structure with ATP, and five protruding mononucleotides at the 50 -terminus hybridized with capture DNA. The 30 -end of the aptamer changed to double-strand DNA due to the target-induced structure-switching. Then Exo III selectivity digested the hairpin DNA and released ATP for analyte recycling, significant signal amplification was achieved. In this way, the target-induced structure-switching aptasensor based on Exo III-catalyzed target recycling for signal amplification was constructed successfully. 2. Experimental
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GE performing as working electrode, a saturated calomel electrode used as reference electrode, and a platinum wire working as auxiliary electrode. The differential pulse voltammetry (DPV) of the proposed aptasensor was scanned from 0.5 to 0 V in PBS (pH 7.4) and the signals were recorded. Fluorescence experiment was performed on RF-540 fluorescence spectrophotometer which was purchased from Shimadzu Ltd. (Japan). 2.3. Preparation of AuNPs and AuNPs–aptamer conjugates AuNPs were prepared by the following method: 250 mL 0.01% HAuCl4 solution was heated to boiling, 4.5 mL of 1% trisodium citrate was added rapidly and kept boiling for 10 min with vigorous stirring condition [22]. Then the prepared AuNPs were naturally cooled to room temperature and stored in opaque glass bottles at 4 C. To synthesize the AuNPs–aptamer conjugates, 500 mL prepared AuNPs was mixed with 20 mL 10 mM SH-DNA, 3 mL 10 mM TCEP. After stirring for 1 h, 50 mL 14.1 mM dATP was added and stirred for 1 h to block the free site of the AuNPs. The solution was centrifuged for 15 min at 12,000 rpm for 3 times, then removed the supernatant and dissolved remaining AuNPs in 500 mL PBS (pH 7.4). 2.4. Fabrication process of the aptasensor The bare gold electrode (GE) was firstly polished with 0.05 mm alumina powder, and cleaned in ethanol and ultrapure water ultrasonically for 5 min. Then the GE was immersed into 0.5 M H2SO4 and scanned from 0 to 1.6 V by cyclic voltammetric (CV) until the peak current of CV became stable. After that, the electrode was dried with N2 and incubated with 1 mM capture DNA containing 5 mM TCEP for 1 h at room temperature. Next, the electrode was immersed into 1 mM MCH for 1 h to seal the left sites and made DNA monolayer well-aligned. Finally, it was soaked into 1 mM hairpin DNA containing 10 U Exo III and differrent concentrations of ATP for 60 min at 37 C. After each modified step, the electrode was washed with PBS (pH 7.4) to remove the nonspecific adsorption.
2.1. Reagents and materials 3. Results and discussion Exo III and 10 NEBuffer 1 were obtained from New England Biolabs, Inc. Adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), uridine triphosphate (UTP) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Hydrogen tetrachloroaurate(III) trihydrate (HAuCl43H2O), trisodium citrate, 6-mercapto-1-hexanol (MCH) and tris(2-carboxyethy) phosphine hydrochloride (TCEP) were ordered from Sigma–Aldrich (USA). All other reagents were analytical reagent. 10 mM Tris–HCl (pH 7.4) contaning 50 mM KCl, 10 mM MgCl2 was used to prepare aptamer and ATP solutions. Oligonucleotides were synthesized by Sangon (Shanghai, China). The sequences of these oligonucleotides were listed as follow: Capture DNA: 50 -GGGGGGATA-SH-30 Hairpin DNA: 50 -MB-ACCCCCATGGAAGGAGGCGTTAGGGGGTCCAT-30 SH-DNA: 50 -MB-ACCCCCATGGAAGGAGGCGTTAGGGGGTCCATSH-30 All aqueous solutions were prepared with ultrapure water (specific resistance >18.2 MV cm1) produced by water purification system (Aquapro).
3.1. Design principle of the proposed aptasensor The electrochemical aptasensor for sensitive ATP detection was based on target-induced structure-switching and Exo III-assisted signal amplification. As shown in Fig. 1, the hairpin DNA consisted of ATP aptamer sequence with methylene blue (MB) labeled at the 50 -terminus, and the 50 -end was six base-pairing to form hairpin structure. Without ATP, the hairpin DNA could not hybridize with the capture DNA, and the 30 -end kept single-strand structure, which resisted the cleavage activity of Exo III. With the addition of ATP, ATP aptamer formed G-quadruplex structure with ATP which resulted in structure-switching of the hairpin DNA. Five protruding mononucleotides at the 50 -end hybridized with capture DNA, and the 30 -end formed double-strand structure. As a result, the MB got close to the electrode surface and showed a strong signal. Then, Exo III selectively digested the double strand from 30 -end, resulting in subsequently the release of ATP. The released ATP again bound with other hairpin DNA to participate in a new hybridization process. The successful Exo III-assisted target recycling process amplified the electrochemical signal significantly.
2.2. Instruments 3.2. Fluorescence experiment All electrochemical measurements were carried out with CHI 660C electrochemical working station (Chenhua, Shanghai). A three-electrode system was used in the experiment with modified
Fluorescence experiment was used to confirm target-induced structure-switching of hairpin DNA. As shown in Fig. 2, MB labeled
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[(Fig._1)TD$IG]
T. Bao et al. / Analytica Chimica Acta 862 (2015) 64–69
Fig. 1. Illustration of the stepwise aptasensor fabrication based on exonuclease III-assisted target recycling for amplification.
To investigate the amplification effect of Exo III, the performance of the proposed aptasensor in the absence and
presence of Exo III were compared. The hairpin DNA (1 mM) was pre-incubated with ATP (10 nM) for 10 min and DPV response of different solutions were recorded. As shown in Fig. 3, DPV signal (curve a) was negligible in the absence of ATP. However, the incubation of ATP with the hairpin DNA led to a small increase in DPV signal (curve b). Such increase was due to the structure-switched hairpin DNA hybridized with capture DNA and MB got close to the electrode surface. When 10 U Exo III was added, a significant increase in DPV signal was observed (curve c), for the Exo III-catalyzed target recycling digested the hairpin DNA circularly, the amount of MB labeled DNA hybridized with capture DNA increased significantly. These results indicated that the Exo III-assisted target recycling amplified the signal significantly.
[(Fig._2)TD$IG] [(Fig._3)TD$IG]
Fig. 2. Fluorescence (FL) emission spectra of the MB in the presence of 2 mM SH-DNA, with (a) 20 nM ATP, no EXO III. (b) AuNPs–SH-DNA conjugates, 20 nM ATP, 10 U Exo III. (c) AuNPs–SH-DNA conjugates, 20 nM ATP, no EXO III.
Fig. 3. Differential pulse voltammograms of aptasensor for 1 mM hairpin DNA after 60 min incubation, with (a) no ATP, no Exo III. (b) 10 nM ATP, no Exo III. (C) 10 nM ATP, 10 U Exo III.
SH-DNA had a strong fluorescent intensity at 692 nm with the excitation wavelength of 665 nm [23] (curve a). As to the AuNPs–aptamer conjugates, AuNPs led to fluorescence quenching of MB, the fluorescence intensity was reduced (curve c). If added in ATP and Exo III, SH-DNA bound to ATP and formed G-quadruplex structure, then Exo III selectively digest the double-strand SH-DNA from its 30 -end. It resulted in the dissociation of G-quadruplex structure and the release of ATP. Hence MB was separated from AuNPs and the fluorescence intensity was restored (curve b). 3.3. Amplification performance of Exo-III assisted target recycling
T. Bao et al. / Analytica Chimica Acta 862 (2015) 64–69
[(Fig._4)TD$IG]
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Fig. 4. EIS (A) and CV (B) for each immobilized step in 0.5 mM K3Fe(CN)6 solution containing 0.1 M KCl: (a) bare GE, (b) capture DNA/GE, (c) MCH/capture DNA/GE, (d) hairpin DNA/ATP//MCH/capture DNA/GE and (d) Exo III/hairpin DNA/ATP/MCH/capture DNA/GE. Inset: the equivalent circuit of EIS.
3.4. Characterization of biosensor fabrication Electrochemical impedance spectroscopy (EIS) and CV measurements were measured to characterize the fabrication of the aptasensor [24]. [Fe(CN)6]3/[Fe(CN)6]4 was used as the redox probe and the semicircle diameter was equal to electron-transfer resistance. In 0.5 mM Fe(CN)63/4, bare GE exhibited an almost straight line (Fig. 4A, curve a), which showed fast electron-transfer process. When capture DNA self-assembled onto the GE, the Ret increased significantly (Fig. 4A, curve b), because the oligonucleotides were non-conductive. After incubated with
MCH, Ret rose continuously (Fig. 4A, curve c). When electrode was incubated with the mixture of hairpin DNA and ATP, Ret increased significantly (Fig. 4A, curve d), for the hairpin DNA hybridized with capture DNA on the electrode. When Exo III was added, Ret further decreased, because of the release of ATP, and the Ret greatly increased (Fig. 4A, curve e), owing to the amount of non-conductive oligonucleotides on the electrode significantly increased through Exo III-assisted target recycling. Insert showed the equivalent circuit of EIS, RS represented the electrolyte resistance, Ret represented the electronic transfer resistance, Cdl represented the double layer capacitance and Zw represented the
[(Fig._5)TD$IG]
Fig. 5. (A) Optimization concentration of capture DNA on the DPV response of the aptasensor. (B) Influence of the dosage of Exo III with 1 mM hairpin DNA, 3 nM ATP. (C) Optimization of incubation time of the aptasensor with the mixture containing 1 mM hairpin DNA, 3 nM ATP and 10 U Exo III.
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T. Bao et al. / Analytica Chimica Acta 862 (2015) 64–69
[(Fig._7)TD$IG]
Warburg impedance. The EIS results were in accordance with CV measurements (Fig. 4B). These results demonstrated the successful fabrication of the aptasensor. 3.5. Optimization of experiment conditions The concentration of capture DNA was an important factor which affected the performance of the aptasensor. In the presence of 3 nM ATP and 0.25 U mL1 Exo III, DPV responses of different concentrations of capture DNA assembled GE were investigated. As shown in Fig. 5A, the DPV signal increased with the increasing concentration of capture DNA and it flattened out at 1 mM. Thus the optimum concentration of capture DNA was chosen as 1 mM. For the optimum concentration of capture DNA was 1 mM, and the hairpin DNA was hybridized with capture DNA at the ratio of 1:1, thus the concentration of hairpin DNA was chosen as 1 mM. And the dosage of Exo III was optimized which had influence on Exo III-assisted target recycling. Different dosages of Exo III were added in the mixture of 1 mM hairpin DNA and 3 nM ATP, the signals were recorded. As shown in Fig. 5B, the DPV response increased with the increase of the dosage of Exo III and it reached a platform at 10 U. Hence the optimum dosage was chosen as 10 U. The incubation time between aptasensor and ATP (with 10 U Exo III) was important to the Exo III-assisted target recycling process. To obtain the optimal incubation time, capture DNA modified electrode was incubated with a mixture containing 1 mM hairpin DNA, 3 nM ATP, and 0.25 U mL1 Exo III at 37 C, and DPV signals at different time were recorded. As is shown in Fig. 5, the signal of DPV increased with the increasing of incubation time and reached a plateau after 60 min. Therefore, 60 min was selected as the optimal incubation time. 3.6. Performance of the proposed aptasensor Under the optimal conditions, the aptasensors were incubated with different concentrations of ATP containing 1 mM hairpin DNA, 0.25 U mL1 Exo III and the DPV responses were recorded. As shown in Fig. 6, the DPV current increased with the concentration of ATP ranging from 0.1 to 20 nM. The corresponding calibration plots were demonstrated in the inset, it was found the increased DPV current exhibited a good liner relationship with the concentration of ATP. The linear equation was fitted as DI (A) = 1.423E 8 + 6.617c (M) (R = 0.9958). The limit of detection (LOD) was 34 pM (S/N = 3),
[(Fig._6)TD$IG]
Fig. 7. Selectivity of the proposed electrochemical biosensor to ATP, GTP, CTP and UTP at 0.1 mM, respectively. Error bars are obtained based on three independent measurements.
which was lower than 9.5 nM [25], which was obtained by fluorescent detection. The specificity of the proposed aptasensor was examined by detecting three interfering agents: GTP, CTP, UTP. The concentration of GTP, CTP, UTP were 0.1 mM. As shown in Fig. 7, three interfering agents led to inconspicuous signal change. However, 0.1 mM ATP caused an obvious increase of DPV signal. It indicated the aptasensor had good selectivity to ATP, due to the specific recognition between ATP aptamer and ATP. To investigate the reproducibility of the proposed aptasensor, the prepared aptasensor were scanned for 100 circles in Tris–HCl (pH 7.4), it was found the current response had no obvious change. In addition, five prepared aptasensors were used to detect 5 nM ATP under the same condition, the relative standard deviation (RSD) of the DPV current was 6.3%, which showed the proposed aptasensor had good reproducibility for ATP detection. 4. Conclusion In summary, we developed a simple and sensitive electrochemical aptasensor for ATP detection based on Exo III-assisted target recycling. The presence of ATP led to structure-switching of the hairpin DNA and created catalytic cleavage sites for Exo III to release ATP for analyte recycling. Exo III-assisted target recycling significantly amplified the signal and greatly improved the sensitivity of the aptasensor. The proposed aptasensor exhibited good selectivity and acceptable reproducibility for ATP detection and had a promising potential for the detection ATP and other small molecules. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 21175032) and the Natural Science Fund for Creative Research Groups of Hubei Province of China (No. 2014CFA015). References
Fig. 6. DPV of 0.1, 0.5, 1, 2.5, 5, 10, 12, 15, 20 nM ATP in PBS (pH 7.4). The inset showed the linear relationship between the increased peak current and the concentration of ATP.
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Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
A sensitive electrochemical aptasensor for ATP detection based on exonuclease III-assisted signal amplification strategy Ting Bao 1, Huawei Shu 1, Wei Wen, Xiuhua Zhang, Shengfu Wang * Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Ministry of Education Key Laboratory for the Synthesis and Application of Organic Functional Molecules & College of Chemistry and Chemical Engineering, Hubei University, Wuhan 430062, PR China
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
Distinctive sequence of hairpinaptamer was designed. Structure-switching of aptamer to reveal the MB labeled terminus. Exonuclease-catalyzed target recycling amplified signal significantly. Exo III improved the sensitivity of the aptasensor significantly.
Illustration of the stepwise aptasensor fabrication based on exonuclease-assisted target recycling for amplification.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 November 2014 Received in revised form 26 December 2014 Accepted 28 December 2014 Available online 30 December 2014
A target-induced structure-switching electrochemical aptasensor for sensitive detection of ATP was successfully constructed which was based on exonuclease III-catalyzed target recycling for signal amplification. With the existence of ATP, methylene blue (MB) labeled hairpin DNA formed G-quadruplex with ATP, which led to conformational changes of the hairpin DNA and created catalytic cleavage sites for exonuclease III (Exo III). Then the structure-switching DNA hybridized with capture DNA which made MB close to electrode surface. Meanwhile, Exo III selectively digested aptamer from its 30 -end, thus G-quadruplex structure was destroyed and ATP was released for target recycling. The Exo III-assisted target recycling amplified electrochemical signal significantly. Fluorescence experiment was performed to confirm the structure-switching process of the hairpin DNA. In fluorescence experiment, AuNPs–aptamer conjugates were synthesized, AuNPs quenched fluorescence of MB, the target-induced structure-switching made Exo III digested aptamer, which restored fluorescence. Under optimized conditions, the proposed aptasensor showed a linear range of 0.1–20 nM with a detection limit of 34 pM. In addition, the proposed aptasensor had good stability and selectivity, offered promising choice for the detection of other small molecules. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Aptamer Electrochemical aptasensor Exonuclease III Signal amplification Adenosine triphosphate
1. Introduction Adenosine triphosphate (ATP) is a multifunctional nucleoside which works as cellular energy currency in living cells [1]. It is
* Corresponding author. Tel.: +86 27 50865309; fax: +86 27 88663043. E-mail address: [email protected] (S. Wang). Both authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.aca.2014.12.049 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.
used as an indicator for cell viability and cell injury and plays a significant role in metabolism and many enzymatic reactions, such as cellular respiration and ions transportation in cells [2,3]. The concentration change of ATP in cells was in connection with many diseases, for instance, angiocardiopathy and Parkinson’s diseases [4,5]. Therefore, sensitive detection of ATP is of great importance in biochemical analysis and clinical diagnoses. Different methods have been used for the detection of ATP, such as ultra performance liquid chromatography [6], fluorescence [7],
T. Bao et al. / Analytica Chimica Acta 862 (2015) 64–69
electrochemiluminescent (ECL) [8] and UV–vis reflectance spectrum [9]. However, limited by their high cost, complex and time-consuming testing process, these methods are not satisfactory. Nevertheless, electrochemical aptasensors have the advantage of low-cost and fast-response, which are proved to be an excellent tool for ATP detection [10]. Aptamers are synthetic single-stranded DNA or RNA which are artificially selected by systematic evolution of ligands by exponential enrichment (SELEX), which could specially recognize diverse targets including protein, metal ions, small molecules and cells [11,12]. ATP aptamer can recognize and bind with ATP to form G-quadruplex structure, it has high affinity and selectivity to ATP [13]. Therefore ATP aptamer is widely used to construct electrochemical aptasensor for sensitive detection of ATP [14,15]. Gold nanoparticles, graphene and exonuclease have been used to amplify the signal of aptasensor. Among them, exonucleasecatalyzed target recycling has been proved to be a perfect method for signal amplification [16–18]. Exonuclease III (Exo III) can selectively digest double-stranded DNA from its 30 -end, which led to the destruction of the aptamer structure and the analyte was released into solution to participate new recycling process [19–21]. In this way, the signal was amplified significantly. Herein, a hairpin DNA consisted of an aptamer sequence for ATP and methylene blue (MB) labeled at the 50 -terminus was ingeniously designed. Exo III could not cleave the hairpin DNA because the 30 -end of hairpin DNA was single-stranded. With the addition of ATP, the hairpin DNA reconfigured to G-quadruplex structure with ATP, and five protruding mononucleotides at the 50 -terminus hybridized with capture DNA. The 30 -end of the aptamer changed to double-strand DNA due to the target-induced structure-switching. Then Exo III selectivity digested the hairpin DNA and released ATP for analyte recycling, significant signal amplification was achieved. In this way, the target-induced structure-switching aptasensor based on Exo III-catalyzed target recycling for signal amplification was constructed successfully. 2. Experimental
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GE performing as working electrode, a saturated calomel electrode used as reference electrode, and a platinum wire working as auxiliary electrode. The differential pulse voltammetry (DPV) of the proposed aptasensor was scanned from 0.5 to 0 V in PBS (pH 7.4) and the signals were recorded. Fluorescence experiment was performed on RF-540 fluorescence spectrophotometer which was purchased from Shimadzu Ltd. (Japan). 2.3. Preparation of AuNPs and AuNPs–aptamer conjugates AuNPs were prepared by the following method: 250 mL 0.01% HAuCl4 solution was heated to boiling, 4.5 mL of 1% trisodium citrate was added rapidly and kept boiling for 10 min with vigorous stirring condition [22]. Then the prepared AuNPs were naturally cooled to room temperature and stored in opaque glass bottles at 4 C. To synthesize the AuNPs–aptamer conjugates, 500 mL prepared AuNPs was mixed with 20 mL 10 mM SH-DNA, 3 mL 10 mM TCEP. After stirring for 1 h, 50 mL 14.1 mM dATP was added and stirred for 1 h to block the free site of the AuNPs. The solution was centrifuged for 15 min at 12,000 rpm for 3 times, then removed the supernatant and dissolved remaining AuNPs in 500 mL PBS (pH 7.4). 2.4. Fabrication process of the aptasensor The bare gold electrode (GE) was firstly polished with 0.05 mm alumina powder, and cleaned in ethanol and ultrapure water ultrasonically for 5 min. Then the GE was immersed into 0.5 M H2SO4 and scanned from 0 to 1.6 V by cyclic voltammetric (CV) until the peak current of CV became stable. After that, the electrode was dried with N2 and incubated with 1 mM capture DNA containing 5 mM TCEP for 1 h at room temperature. Next, the electrode was immersed into 1 mM MCH for 1 h to seal the left sites and made DNA monolayer well-aligned. Finally, it was soaked into 1 mM hairpin DNA containing 10 U Exo III and differrent concentrations of ATP for 60 min at 37 C. After each modified step, the electrode was washed with PBS (pH 7.4) to remove the nonspecific adsorption.
2.1. Reagents and materials 3. Results and discussion Exo III and 10 NEBuffer 1 were obtained from New England Biolabs, Inc. Adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), uridine triphosphate (UTP) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Hydrogen tetrachloroaurate(III) trihydrate (HAuCl43H2O), trisodium citrate, 6-mercapto-1-hexanol (MCH) and tris(2-carboxyethy) phosphine hydrochloride (TCEP) were ordered from Sigma–Aldrich (USA). All other reagents were analytical reagent. 10 mM Tris–HCl (pH 7.4) contaning 50 mM KCl, 10 mM MgCl2 was used to prepare aptamer and ATP solutions. Oligonucleotides were synthesized by Sangon (Shanghai, China). The sequences of these oligonucleotides were listed as follow: Capture DNA: 50 -GGGGGGATA-SH-30 Hairpin DNA: 50 -MB-ACCCCCATGGAAGGAGGCGTTAGGGGGTCCAT-30 SH-DNA: 50 -MB-ACCCCCATGGAAGGAGGCGTTAGGGGGTCCATSH-30 All aqueous solutions were prepared with ultrapure water (specific resistance >18.2 MV cm1) produced by water purification system (Aquapro).
3.1. Design principle of the proposed aptasensor The electrochemical aptasensor for sensitive ATP detection was based on target-induced structure-switching and Exo III-assisted signal amplification. As shown in Fig. 1, the hairpin DNA consisted of ATP aptamer sequence with methylene blue (MB) labeled at the 50 -terminus, and the 50 -end was six base-pairing to form hairpin structure. Without ATP, the hairpin DNA could not hybridize with the capture DNA, and the 30 -end kept single-strand structure, which resisted the cleavage activity of Exo III. With the addition of ATP, ATP aptamer formed G-quadruplex structure with ATP which resulted in structure-switching of the hairpin DNA. Five protruding mononucleotides at the 50 -end hybridized with capture DNA, and the 30 -end formed double-strand structure. As a result, the MB got close to the electrode surface and showed a strong signal. Then, Exo III selectively digested the double strand from 30 -end, resulting in subsequently the release of ATP. The released ATP again bound with other hairpin DNA to participate in a new hybridization process. The successful Exo III-assisted target recycling process amplified the electrochemical signal significantly.
2.2. Instruments 3.2. Fluorescence experiment All electrochemical measurements were carried out with CHI 660C electrochemical working station (Chenhua, Shanghai). A three-electrode system was used in the experiment with modified
Fluorescence experiment was used to confirm target-induced structure-switching of hairpin DNA. As shown in Fig. 2, MB labeled
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Fig. 1. Illustration of the stepwise aptasensor fabrication based on exonuclease III-assisted target recycling for amplification.
To investigate the amplification effect of Exo III, the performance of the proposed aptasensor in the absence and
presence of Exo III were compared. The hairpin DNA (1 mM) was pre-incubated with ATP (10 nM) for 10 min and DPV response of different solutions were recorded. As shown in Fig. 3, DPV signal (curve a) was negligible in the absence of ATP. However, the incubation of ATP with the hairpin DNA led to a small increase in DPV signal (curve b). Such increase was due to the structure-switched hairpin DNA hybridized with capture DNA and MB got close to the electrode surface. When 10 U Exo III was added, a significant increase in DPV signal was observed (curve c), for the Exo III-catalyzed target recycling digested the hairpin DNA circularly, the amount of MB labeled DNA hybridized with capture DNA increased significantly. These results indicated that the Exo III-assisted target recycling amplified the signal significantly.
[(Fig._2)TD$IG] [(Fig._3)TD$IG]
Fig. 2. Fluorescence (FL) emission spectra of the MB in the presence of 2 mM SH-DNA, with (a) 20 nM ATP, no EXO III. (b) AuNPs–SH-DNA conjugates, 20 nM ATP, 10 U Exo III. (c) AuNPs–SH-DNA conjugates, 20 nM ATP, no EXO III.
Fig. 3. Differential pulse voltammograms of aptasensor for 1 mM hairpin DNA after 60 min incubation, with (a) no ATP, no Exo III. (b) 10 nM ATP, no Exo III. (C) 10 nM ATP, 10 U Exo III.
SH-DNA had a strong fluorescent intensity at 692 nm with the excitation wavelength of 665 nm [23] (curve a). As to the AuNPs–aptamer conjugates, AuNPs led to fluorescence quenching of MB, the fluorescence intensity was reduced (curve c). If added in ATP and Exo III, SH-DNA bound to ATP and formed G-quadruplex structure, then Exo III selectively digest the double-strand SH-DNA from its 30 -end. It resulted in the dissociation of G-quadruplex structure and the release of ATP. Hence MB was separated from AuNPs and the fluorescence intensity was restored (curve b). 3.3. Amplification performance of Exo-III assisted target recycling
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[(Fig._4)TD$IG]
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Fig. 4. EIS (A) and CV (B) for each immobilized step in 0.5 mM K3Fe(CN)6 solution containing 0.1 M KCl: (a) bare GE, (b) capture DNA/GE, (c) MCH/capture DNA/GE, (d) hairpin DNA/ATP//MCH/capture DNA/GE and (d) Exo III/hairpin DNA/ATP/MCH/capture DNA/GE. Inset: the equivalent circuit of EIS.
3.4. Characterization of biosensor fabrication Electrochemical impedance spectroscopy (EIS) and CV measurements were measured to characterize the fabrication of the aptasensor [24]. [Fe(CN)6]3/[Fe(CN)6]4 was used as the redox probe and the semicircle diameter was equal to electron-transfer resistance. In 0.5 mM Fe(CN)63/4, bare GE exhibited an almost straight line (Fig. 4A, curve a), which showed fast electron-transfer process. When capture DNA self-assembled onto the GE, the Ret increased significantly (Fig. 4A, curve b), because the oligonucleotides were non-conductive. After incubated with
MCH, Ret rose continuously (Fig. 4A, curve c). When electrode was incubated with the mixture of hairpin DNA and ATP, Ret increased significantly (Fig. 4A, curve d), for the hairpin DNA hybridized with capture DNA on the electrode. When Exo III was added, Ret further decreased, because of the release of ATP, and the Ret greatly increased (Fig. 4A, curve e), owing to the amount of non-conductive oligonucleotides on the electrode significantly increased through Exo III-assisted target recycling. Insert showed the equivalent circuit of EIS, RS represented the electrolyte resistance, Ret represented the electronic transfer resistance, Cdl represented the double layer capacitance and Zw represented the
[(Fig._5)TD$IG]
Fig. 5. (A) Optimization concentration of capture DNA on the DPV response of the aptasensor. (B) Influence of the dosage of Exo III with 1 mM hairpin DNA, 3 nM ATP. (C) Optimization of incubation time of the aptasensor with the mixture containing 1 mM hairpin DNA, 3 nM ATP and 10 U Exo III.
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[(Fig._7)TD$IG]
Warburg impedance. The EIS results were in accordance with CV measurements (Fig. 4B). These results demonstrated the successful fabrication of the aptasensor. 3.5. Optimization of experiment conditions The concentration of capture DNA was an important factor which affected the performance of the aptasensor. In the presence of 3 nM ATP and 0.25 U mL1 Exo III, DPV responses of different concentrations of capture DNA assembled GE were investigated. As shown in Fig. 5A, the DPV signal increased with the increasing concentration of capture DNA and it flattened out at 1 mM. Thus the optimum concentration of capture DNA was chosen as 1 mM. For the optimum concentration of capture DNA was 1 mM, and the hairpin DNA was hybridized with capture DNA at the ratio of 1:1, thus the concentration of hairpin DNA was chosen as 1 mM. And the dosage of Exo III was optimized which had influence on Exo III-assisted target recycling. Different dosages of Exo III were added in the mixture of 1 mM hairpin DNA and 3 nM ATP, the signals were recorded. As shown in Fig. 5B, the DPV response increased with the increase of the dosage of Exo III and it reached a platform at 10 U. Hence the optimum dosage was chosen as 10 U. The incubation time between aptasensor and ATP (with 10 U Exo III) was important to the Exo III-assisted target recycling process. To obtain the optimal incubation time, capture DNA modified electrode was incubated with a mixture containing 1 mM hairpin DNA, 3 nM ATP, and 0.25 U mL1 Exo III at 37 C, and DPV signals at different time were recorded. As is shown in Fig. 5, the signal of DPV increased with the increasing of incubation time and reached a plateau after 60 min. Therefore, 60 min was selected as the optimal incubation time. 3.6. Performance of the proposed aptasensor Under the optimal conditions, the aptasensors were incubated with different concentrations of ATP containing 1 mM hairpin DNA, 0.25 U mL1 Exo III and the DPV responses were recorded. As shown in Fig. 6, the DPV current increased with the concentration of ATP ranging from 0.1 to 20 nM. The corresponding calibration plots were demonstrated in the inset, it was found the increased DPV current exhibited a good liner relationship with the concentration of ATP. The linear equation was fitted as DI (A) = 1.423E 8 + 6.617c (M) (R = 0.9958). The limit of detection (LOD) was 34 pM (S/N = 3),
[(Fig._6)TD$IG]
Fig. 7. Selectivity of the proposed electrochemical biosensor to ATP, GTP, CTP and UTP at 0.1 mM, respectively. Error bars are obtained based on three independent measurements.
which was lower than 9.5 nM [25], which was obtained by fluorescent detection. The specificity of the proposed aptasensor was examined by detecting three interfering agents: GTP, CTP, UTP. The concentration of GTP, CTP, UTP were 0.1 mM. As shown in Fig. 7, three interfering agents led to inconspicuous signal change. However, 0.1 mM ATP caused an obvious increase of DPV signal. It indicated the aptasensor had good selectivity to ATP, due to the specific recognition between ATP aptamer and ATP. To investigate the reproducibility of the proposed aptasensor, the prepared aptasensor were scanned for 100 circles in Tris–HCl (pH 7.4), it was found the current response had no obvious change. In addition, five prepared aptasensors were used to detect 5 nM ATP under the same condition, the relative standard deviation (RSD) of the DPV current was 6.3%, which showed the proposed aptasensor had good reproducibility for ATP detection. 4. Conclusion In summary, we developed a simple and sensitive electrochemical aptasensor for ATP detection based on Exo III-assisted target recycling. The presence of ATP led to structure-switching of the hairpin DNA and created catalytic cleavage sites for Exo III to release ATP for analyte recycling. Exo III-assisted target recycling significantly amplified the signal and greatly improved the sensitivity of the aptasensor. The proposed aptasensor exhibited good selectivity and acceptable reproducibility for ATP detection and had a promising potential for the detection ATP and other small molecules. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 21175032) and the Natural Science Fund for Creative Research Groups of Hubei Province of China (No. 2014CFA015). References
Fig. 6. DPV of 0.1, 0.5, 1, 2.5, 5, 10, 12, 15, 20 nM ATP in PBS (pH 7.4). The inset showed the linear relationship between the increased peak current and the concentration of ATP.
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