Talanta 78 (2009) 954–958
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A sensitive, label free electrochemical aptasensor for ATP detection Wang Li, Zhou Nie ∗ , Xiahong Xu, Qinpeng Shen, Chunyan Deng, Jinhua Chen ∗ , Shouzhuo Yao State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, PR China
a r t i c l e
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Article history: Received 3 October 2008 Received in revised form 4 January 2009 Accepted 5 January 2009 Available online 20 January 2009 Keywords: ATP detection Aptasensor Gold nanoparticles Chronocoulometry
a b s t r a c t A sensitive, label free electrochemical aptasensor for small molecular detection has been developed in this work based on gold nanoparticles (AuNPs) amplification. This aptasensor was fabricated as a tertiary hybrid DNA–AuNPs system, which involved the anchored DNA (ADNA) immobilized on gold electrode, reporter DNA (RDNA) tethered with AuNPs and target-responsive DNA (TRDNA) linking ADNA and RDNA. Electrochemical signal is derived from chronocoulometric interrogation of [Ru(NH3 )6 ]3+ (RuHex) that quantitatively binds to surface-confined DNA via electrostatic interaction. Using adenosine triphosphate (ATP) as a model analyte and ATP-binding aptamer as a model molecular reorganization element, the introduction of ATP triggers the structure switching of the TRDNA to form aptamer–ATP complex, which results in the dissociation of the RDNA capped AuNPs (RDNA–AuNPs) and release of abundant RuHex molecules trapped by RDNA–AuNPs. The incorporation of AuNPs in this strategy significantly enhances the sensitivity because of the amplification of electrochemical signal by the RDNA–AuNPs/RuHex system. Under optimized conditions, a wide linear dynamic range of 4 orders of magnitude (1 nM–10 M) was reached with the minimum detectable concentration at sub-nanomolar level (0.2 nM). Those results demonstrate that our nanoparticles-based amplification strategy is feasible for ATP assay and presents a potential universal method for other small molecular aptasensors. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Detecting and quantifying small molecular substances such as drugs, hormones and adenosine phosphorylated derivatives are increasingly needed in environmental analysis and clinical assay [1–4]. Great progress has been achieved in the development of analytical assays of small molecules with different detection techniques (optical [5], electrochemical [6], piezoelectric [7], electrochemiluminescent (ECL) [8], chromatographic [9], surface-enhanced Raman scattering (SERS) [10], etc.). Among them, electrochemical detection methods became a popular technology because of their numerous merits, including high sensitivity, simple instrumentation, low production cost, fast response and portability [11,12]. Electrochemical affinity sensors, an important part of electrochemical sensors, commonly rely on the immobilization of a biological recognition element (antibodies or aptamers) onto the transducer surface. In recent years, electrochemical affinity sensors for the detection of small molecules, especially non-redox small molecules, have attracted much attention because of good affinity to their target elements [7,12,13]. Aptamers [14,15] are single-stranded DNA or RNA sequences artificially selected through systematic evolution of ligands by
∗ Corresponding authors. Tel.: +86 731 8821961; fax: +86 731 8821848. E-mail addresses:
[email protected] (Z. Nie),
[email protected] (J. Chen). 0039-9140/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2009.01.009
exponential enrichment (SELEX). They are used to bind various targets, such as small molecules [12,16], proteins [6,17] and even viruses and cells [18], with high specificity and affinity. In despite of the similar identification principle for targets with antibodies, aptamers can provide several advantages over antibodies such as simple synthesis, easy labeling, good stability, wide applicability, and high sensitivity. In addition, aptamers are readily applicable to the identification of small molecules that make them superior to antibodies when used as sensing elements for small molecular sensors [19]. Antibodies are applied hardly and complicatedly to the small molecules which are toxic to the host animal or trigger a minimal immunogenic response [19], whereas aptamers could be applied to various small molecules with no limitation. The nucleic acid nature and intrinsic advantages of aptamers, integrated with the previously well-developed nucleic acid sensor technologies, promoted small molecular aptasensor technologies growing rapidly in recent years [18–21]. Although aptamers are the promising small molecular recognition elements, a major disadvantage of aptamers is their relatively low association constants with the small molecules, which leads to a rather poor detection limit. Due to this drawback, the high sensitivity requested in aptamer-based assay for the detection of small molecules is hardly to be reached by simple “direct” binding protocols. Therefore, developing effective amplification paths for small molecular aptasensor is important. With this aim, several methods have been employed to amplify the signal, such as DNAzyme
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[22], rolling cycle amplification (RCA) [23], and strand displacement amplification (SDA) [24]. However, the sensitivity of these amplification methods was still not satisfactory (millimolar level). These methods might be hampered by the relatively low catalytic activity of DNA peroxidase or suffer from high cost, time-consuming and complex reaction system for the nucleic acid amplification (RCA or SDA). In recent years, taking the advantage of their high stability, low cost, and labeling convenience, inorganic nanoparticles were employed as signal amplification elements in various DNA or protein electrochemical assays [25–27]. Using gold nanoparticles (AuNPs) as the signal amplification elements, Fan and co-workers [27] reported a novel inorganic nanoparticle-based electrochemical DNA sensor that could sensitively detect femtomolar target DNA. The electrochemical signals of this sensor were generated by chronocoulometric interrogation of [Ru(NH3 )6 ]3+ (RuHex) through electrostatic adsorption, which was better than labeled methods. In this paper, this sensitive and label free electrochemical strategy is extended to the small molecular detection and is used to detect adenosine triphosphate (ATP). It is well-known that ATP is the mediator of energy exchanges that occur in all living cells, in both catabolic and anabolic processes and is widely used as an index for biomass determinations in clinical microbiology, food quality control and environmental analyses [4,28]. Therefore, in this work, it is chosen as a model analyte and ATP-binding aptamer is taken as a model molecular reorganization element. The proposed ATP aptasensor is based on the DNA–AuNPs hybrid system containing three functional components: AuNPs functionalized with 5 -thiol-modified reporter DNA (RDNA), 3 -thiol-modified anchored DNA (ADNA) immobilized on electrode, and a target-responsive DNA (TRDNA). Here, TRDNA acts as both the recognized element specific for ATP and the linker connecting RDNA and ADNA. The TRDNA is composed of three segments: first, the segment (12 nucleotides) hybridized with ADNA, second, the segment hybridized with the last five nucleotides of RDNA, and third, the segment (the aptamer sequence for ATP) hybridized with the other seven nucleotides on the RDNA. When an aptamer immobilized on the electrode surface captures two ATP molecules, the binding-induced conformational changes lead to the release of the DNA–AuNPs as well as the numerous binding RuHex molecules, resulting in the transduction and amplification of the ATP-binding signal. Based on this strategy, we found this sensor can sensitively detect sub-nanomolar ATP, and provide a universal and sensitive method for small molecular assay. 2. Experimental 2.1. Materials and apparatus All oligonucleotides were synthesized and purified by Sangon Inc. (Shanghai, China). The sequences of the single-stranded oligonucleotides are as follows:
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prepared with Milli-Q water (18.25 M cm) from a Millipore system. Electrochemical measurements were performed with a CHI 660a electrochemical workstation (Shanghai Chenhua Instrument Corporation, China). A conventional three-electrode cell was employed, which involved a gold working electrode (2 mm in diameter), a platinum foil counter electrode, and a saturated calomel reference electrode (SCE). All the potentials in this paper were with respect to SCE. The electrolyte buffer was thoroughly purged with nitrogen before experiments. 2.2. Functionalization of AuNPs with reporter DNA The RDNA functionalized AuNPs (RDNA–AuNPs) were prepared as reported in the literature [27]. Briefly, RDNA–AuNPs were synthesized by incubating RDNA (3.0 M) in 1 mL of 13 nm AuNPs solution (1.2 nM) for 16 h. Then, the RDNA–AuNPs conjugates were “aged” in salt condition (0.1 M NaCl) for 40 h. Excess reagents were removed by centrifuging at 15,000 rpm for 30 min. The red precipitate was washed, re-centrifuged, and then dispersed in 1 mL solution (0.25 M NaCl, 10 mM phosphate buffer, pH 7.0). 2.3. Preparation of the aptasensor and ATP detection Gold electrode was polished sequentially with 0.3 and 0.05 m alumina powder followed by ultrasonic cleaning with distilled water, ethanol, and distilled water for 5 min each. Then the electrode was electrochemically cleaned to remove any remaining impurities. Finally, the electrode was washed with distilled water and dried in a mild nitrogen stream. RDNA–AuNPs/TRDNA/ADNA modified gold electrode was earned by placing 4 L of the ADNA solution (10 mM Tris–HCl, 1 mM EDTA, 10 mM TCEP, and 0.1 M NaCl (pH 7.4)) on gold electrode, and further treated with 1 mM MCH for 2 h to obtain well aligned DNA monolayers. Then, 4 L of 10 M TRDNA solution was placed on electrode for 2 h. At last 4 L of RDNA–AuNPs was dropped on the electrode for 2 h. For the ATP detection, 4 L of ATP solution (a series of concentrations from 0.2 × 10−9 to 1.0 × 10−3 M) was placed onto the modified electrode. Each process was followed with washing and drying. Chronocoulometry (CC) was carried out in Tris buffer (10 mM Tris–HCl, pH 7.4) containing 50 M RuHex. The surface density of DNA ( ss ) was measured with CC as described in literature [30], ss = [(Qtotal − Qdl )NA /nFA](z/m). Where Qtotal stands for the total charge flowing through the electrode, comprising both Faradaic (redox) and non-Faradaic (capacitive) charges; Qdl the capacitive charge; n the number of electrons in the reaction; A the surface area of the working electrode; m the number of nucleotides in the DNA; z the charge of the redox molecules and NA the Avogadro’s number. 2.4. Preparation of urine samples
ADNA: 5 -TCA CAG ATG AGT TT-SH-3 RDNA: 5 -HS-CCC AGG TTC TCT-3 TRDNA: 5 -ACT CAT CTG TGA AGA GAA CCT GGG GGA GTA TTG CGG AGG AAG GT-3 AuNPs were synthesized according to the published protocol [29]. Mercaptohexanol (MCH), hexaamineruthenium(III) chloride ([Ru(NH3 )6 ]3+ , RuHex), and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were purchased from Sigma (St. Louis, MO). ATP, GTP, UTP and CTP were from BBI (Canada). Tris(hydroxymethyl)aminomethane (Tris) was obtained from Oumay Biotech Co. Ltd. (Changsha, China). All the reagents mentioned above were used without further purification. All solutions were
Fresh urine samples were obtained from healthy volunteers. Each sample was filtered through a 0.2-mm membrane to remove particulate matter. The human-urine samples were diluted separately by a factor of 100 with the buffer solution and then were equilibrated for 30 min at room temperature. 3. Results and discussion 3.1. Design strategy of the nanoparticles-based ATP aptasensor In this work, the electroactive complex, RuHex, serves as the signal molecule, since the RuHex cations can associate with anionic
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panied with the extrication of the abundant RuHex molecules, which offers a significant amplification in the capture event. The design relies on the structure-switching properties of aptamers upon binding to their target molecules. Because there are no special requirements on the aptamer part, this strategy is generally applicable to many small molecular aptamers. 3.2. Characterization of the sensor
Fig. 1. The representation of the aptasensor based on nanoparticles amplification for ATP detection. The color of DNA: ADNA–green, TRDNA–black, RDNA–red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
phosphates of DNA strands as counterions [27]. It has been demonstrated that binding of RuHex to DNA is totally through electrostatic interaction with intrinsic stoichiometric ratio. Thus redox charge of the trapped RuHex is a direct function of the amount of the DNA strands confined to the electrode surface [30]. Based on this strategy, DNA functionalized AuNPs, loaded with hundreds of DNA strands per particle, are used as the signal amplification element in electrochemical assay by absorbing thousands of RuHex molecules. Compared with the covalent redox-labeling technique broadly used in existing electrochemical aptasensors [12,16], the redox probe (RuHex) electrostatic binding strategy is relatively convenient, time-saving and without requirement of purification. Fig. 1 shows the principle of the nanoparticles-based electrochemical ATP aptasensor for ATP detection. In the absence of ATP, ADNA and RDNA are assembled with TRDNA to form tertiary complex, which brings the RDNA–AuNPs proximate to the electrode surface. The RDNA–AuNPs localized at electrode surface can electrostatically absorb abundant redox charge of numerous RuHex molecules close to the electrode surface. When the ATP is introduced, the aptamer switches its structure to bind ATP and prefers to form an ATP–aptamer complex rather than an aptamer–DNA duplex [31]. As a result, only five base pairs are left to hybridize with RDNA, which is unstable at room temperature [32] and results in the dissociation of RDNA–AuNPs. The release of RDNA–AuNPs is accom-
Electrochemical impedance spectroscopy (EIS) was employed for the electrochemical characterization of the modified electrode in 10 mM [Fe(CN)6 ]4−/3− and 0.1 M KCl aqueous solution and the corresponding results are shown in Fig. 2(A). The electron-transfer resistance increases in the order of the bare electrode (a), TRDNA/ADNA modified electrode (b) and RDNA–AuNPs/TRDNA/ADNA modified electrode (c). Because the negative charge of DNA hinders [Fe(CN)6 ]4−/3− from reaching to the electrode surface, and the change of resistance reflects the amount of immobilized DNA, the increase in electron-transfer resistance indicates that the TRDNA and RDNA–AuNPs are successfully immobilized on the electrode surface. After the electrode was treated with 1 M ATP (d), the electron-transfer resistance of the electrode decreases obviously (from 1.7 k to 1 k). This result demonstrates that the aptamer sequences were successfully attached with ATP and DNA–AuNPs were dissociated from the surface of the electrode. On the other hand, differential pulse voltammetry (DPV) was employed to characterize the electrochemistry of RuHex. As shown in Fig. 2(B), two peaks (peaks I and II) can be observed when the DNA/MCH modified electrode (TRDNA/ADNA or RDNA–AuNPs/TRDNA/ADNA modified gold electrode) is immersed in a solution containing RuHex at a low ionic strength. Peak I, observed for the DNA/MCH modified electrode and also the MCH only modified electrode (data not shown), should be ascribed to the diffusion-based redox process of RuHex (RuHex diffused to the electrode). The other peak (peak II), observed at about −0.34 V, is due to the surface-confined redox process of RuHex electrostatically bound to the phosphate backbone of DNA [27]. Since the surface-confined redox signal can reflect the amount of DNA strands located at the electrode surface, the RDNA–AuNPs loaded on electrode surface can be definitely indicated by the change of the current intensity of peak II. It is noted that a small peak current (peak II) is observed at the TRDNA/ADNA electrode (a), and a significant enhancement of peak II can be found after the formation of the RDNA–AuNPs/TRDNA/ADNA complex (b). This suggests that the On/Off switch of the localization of RDNA–AuNPs on electrode
Fig. 2. Characterization of the aptasensor: (A) The electrochemical impedance spectra of bare (a), TRDNA/ADNA modified (b) and RDNA–AuNPs/TRDNA/ADNA modified (c) gold electrodes in 10 mM [Fe(CN)6 ]4−/3− and 0.1 M KCl aqueous solution. (d) is (c) treated with 1 M of ATP. The frequency changed from 0.1 Hz to 100,000 Hz and the amplitude was 5.0 mV. (B) Differential pulse voltammograms of the (a) TRDNA/ADNA modified and (b) RDNA–AuNPs/TRDNA/ADNA modified gold electrodes in 10 mM Tris buffer containing 50 M RuHex.
W. Li et al. / Talanta 78 (2009) 954–958 Table 1 The effect of the surface density of ADNA on the detection efficiency of the electrode. The standard deviations of measurements were taken from four independent experiments. ADNA concentration (M)
Surface density of ADNA (molecule/cm2 )
Q (C)
5 1 0.2 0.04 0.02
4.0 × 1012 2.3 × 1012 1.0 × 1012 4.9 × 1011 3.9 × 1011
0.16 0.24 0.38 0.21 0.10
± ± ± ± ±
0.01 0.01 0.03 0.02 0.02
surface can remarkably amplify the variation of surface-confined redox signal. Fan and co-workers have demonstrated that chronocoulometry is more accurate for detecting the signal of electrostatically trapped redox marker than other electrochemical methods, and RuHex–DNA–electrode system can be used to generate an intense signal in CC [33]. Hence, the CC technique was employed for the subsequent ATP detection experiments. 3.3. Optimization of surface density of anchored DNA for effective ATP detection In order to obtain a perfect sensitivity, an appropriate surface density of the immobilized ADNA is needed. In this work, five kinds of ADNA self-assembled monolayers (SAMs) with different surface density of ADNA (4.0 × 1012 , 2.3 × 1012 , 1.0 × 1012 , 4.9 × 1011 and 3.9 × 1011 molecule/cm2 ) were prepared by placing ADNA solution with different concentration (5.0, 1.0, 0.2, 0.04 and 0.02 M) in the same self-assemble time (60 min). After the preparation of ADNA SAMs, TRDNA and RDNA–AuNPs were assembled on the ADNA SAMs successively in sufficient hybridization time. The detection efficiency of the resulting electrodes was investigated in the presence of 10 M ATP. As shown in Table 1, the largest CC signal change (Q = Qtotal − Qdl ) occurred at the surface density of ADNA 1.0 × 1012 molecule/cm2 . The CC signal change increases with the decrease of the surface density of ADNA when the density is larger than 1.0 × 1012 molecule/cm2 . This phenomenon is probably caused by the too dense ADNA–TRDNA on electrode that cannot fully hybrid with RDNA–AuNPs. The ADNA–TRDNA without RDNA–AuNP hybridization could lead to lose the signal amplification after ATP catching, resulting in sensitivity decrease. However, the CC signal change decreases with the decrease of the surface density of ADNA when the density of ADNA is less than 1.0 × 1012 molecule/cm2 . It is probably because the ADNA–TRDNAs can fully
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hybrid with RDNA–AuNPs when the surface density of ADNA is less than 1.0 × 1012 molecule/cm2 . The high surface density of the ADNA–TRDNA–RDNA–AuNP element could increase the efficiency of ATP capture and the sensitivity of the sensor. Therefore, this clearly shows that control of DNA assembly density is essential for the improvement of sensitivity, and the optimized DNA monolayer (1.0 × 1012 molecule/cm2 ) was adopted in the following experiments. 3.4. Detection of ATP The detection performance of the ATP sensor was evaluated by exposing the sensor to a series of ATP concentrations (from 0.2 nM to 1.0 mM) under the same experimental condition. As shown in Fig. 3(A), the increase in ATP concentration induces a monotonous decrease in CC signal (Q). The plot of the CC signal as a function of ATP concentrations is illustrated in Fig. 3(B). It is found that the value of Q is logarithmically related to the ATP concentration. The linear range is from 1 nM to 10 M. The calibration equation was Q = 0.0808 log C + 0.0558 with a correlation coefficient of 0.993. The minimum detectable ATP concentration is at sub-nanomolar level (0.2 nM). The results indicate that the present method can successfully detect the ATP with high sensitivity and low detection limit. Recently, Shao and co-workers reported an electrochemical approach for ATP detection by using the similar CC protocol but without amplification by nanoparticles [21]. They reported that the detection range was from 0.1 M to 1 mM and detection limit is below 0.1 M, which are about 2 orders of magnitude higher than those of our method. The high sensitivity of our sensor is contributed to the presence of AuNPs. In our amplification strategy, gold nanoparticle loaded with a few hundred DNA strands was introduced instead of only one complementary DNA strand as leaving group responded in the aptamer–ATP-binding event. It is worth to note that the detection limit of this aptasensor with nanoparticlesbased amplification is more than 3 orders of magnitude lower than that of the aptasensor based on nucleic acid amplification strategy. The sub-nanomolar sensitivity of the present sensor is much better than other ATP aptasensors reported in the literatures, including luminescent [34], electrochemical [12,21], SERS [10] and colorimetrical [5] aptasensors. 3.5. Selectivity and nonspecific adsorption of the sensor Besides sensitivity, selectivity is also an important feature for biosensors. CTP, UTP, and GTP, which belong to the nucleoside triphosphate family, are usually coexisting with ATP in real biolog-
Fig. 3. (A) Chronocoulometry curves for electrodes exposed to ATP solution with a series of concentrations (from a to g: 1 nM, 10 nM, 100 nM, 1 M, 10 M, 100 M and 1 mM). The electrolyte is 10 mM Tris buffer containing 50 M RuHex. Pulse period: 250 ms; pulse width: 700 mV. Intercepts at t = 0 in chronocoulometric curves represent redox charges of RuHex bound to DNA. (B) Calibration curve for the detection of ATP. Error bars show the standard deviations of measurements taken from four independent experiments.
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4. Conclusion
Fig. 4. The selectivity of the aptasensor. The sensor was treated with 10 M (ATP, CTP, UTP, or GTP) sample solution for 60 min. Other conditions are the same as those described in Fig. 3(A). Error bars show the standard deviations of measurements taken from four independent experiments.
We developed a novel, label free small molecular chronocoulometric aptasensor based on gold nanoparticles signal-amplified mechanism. DNA capped gold nanoparticles are demonstrated as potent amplifier for the aptamer–analyte binding event, which causes significant chronocoulometric signal change of surfaceconfined RuHex. As a model system, ATP was chosen as the model analyte and ATP-binding aptamer was taken as the model molecular reorganization element. This aptasensor can detect as low as sub-nanomolar ATP, which is 3 orders of magnitude more sensitive than the existing aptasensors based on nucleic acid amplification. Furthermore, this novel ATP sensor showed fairly good selectivity. This aptamer sensing strategy based on nanoparticles amplification is versatile and has great potential in the construction of aptamerbased biosensors for the detection of various small molecules. Acknowledgments
Table 2 Recovery of ATP assays in real biological samples. The standard deviations of measurements were taken from four independent experiments. Sample
Added (nM)
Found (nM)
1 2 3 4 5
45.5 200 500 2000 5000
51.8 190 440 1816 4650
± ± ± ± ±
8.1 23 54 225 660
Recovery 114% 95% 88% 91% 93%
ical samples. Differentiation of the other nucleoside triphosphate from ATP is of significant importance for sensing ATP in biochemical assay. Fig. 4 exhibits that only small signal changes took place after the addition of 4 L of 10 M GTP, UTP, or CTP compared to the addition of 4 L of 10 M ATP. It was found that the Q for 10 M GTP (UTP, or CTP) equals to that for 1 nM ATP. Furthermore, the Q for 1 mM GTP (UTP, or CTP) equals to that for 4 nM ATP (not shown in the figure). This indicates that the proposed strategy has sufficient selectivity in ATP detection, and is able to discriminate ATP in complex samples from its analogues. The excellent selectivity of the sensor arises not only from the high selectivity of ATP aptamer but also from the additional stringency due to the competition between the aptamer-complementary strand duplex and the aptamer–ATP structure. It was reported that the nonspecific binding of DNA-conjugated AuNPs to solid surfaces is much more severe than that of DNA [35]. Therefore, we investigated the nonspecific adsorption of RDNA–AuNPs onto the electrode surface by dropping 4 L of RDNA–AuNPs (1.2 nM) onto the ADNA modified electrode for 2 h. We found that a small amount of AuNPs could be nonspecifically adsorbed onto the electrode surface and the respective signal arising was only 18.3 ± 6.6 nC, which was much less than the signal of 0.2 nM ATP (Q = 33 ± 8 nC). 3.6. Recovery test The recovery experiment of different ATP concentrations was carried out to evaluate applicability and reliability of the developed electrochemical aptasensors in complex system. Urine samples were employed in this work as the model complex system. Three ATP added samples were prepared and the results are shown in Table 2. The recoveries for the added ATP with 45.5 nM, 200 nM, 500 nM, 2 M, and 5 M are 114%, 95%, 88%, 91%, and 93%, respectively.
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