Adenosine Triphosphate Sensing by Electrocatalysis with DNAzyme

DOI: 10.1002/elan.201300425

Adenosine Triphosphate Sensing by Electrocatalysis withDNAzymeGuangfeng Wang,*[a] Ling Chen,[a] Yanhong Zhu,[a] Lun Wang,[a] and Xiaojun Zhang*[a]

1 Introduction

DNAzymes are a kind of artificial enzymes which exhibitspecific catalytic activities [1]. Since the first deoxyribo-zyme (catalytic DNAzymes) was discovered in 1994 [2], ithas been seen significant progress in studying DNAzyme[3–5]. Compared with natural proteinogenic enzymes,DNAzymes are easier to synthesize and modify, less ex-pensive and with higher thermal stability. Besides, theflexibility in mastering the DNAzyme structures by en-coding recognition function into DNAzyme sequencesmakes DNAzymes ideal candidates for developing bio-analytical platforms. Peroxidase-like G-quadruplex-hemincomplexes, (HRP-DNAzyme, G-quadruplex-based DNA-zyme), a class of important DNAzymes, were first report-ed by Travascio et al [6, 7]. It is formed by hemin anda G-quadruplex aptamer which can catalyze H2O2-medi-ated oxidation of 2, 2’-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), luminol or H2DCFDA [2, 2’-azino-bis (3-ethylben-zthiazoline-6-sulfonic acid)], provid-ing means for the optical readout of the biosensing pro-cesses [7, 8]. Compared with other DNAzymes, G-quadru-plex-based DNAzyme has the advantages of low produc-tion cost, easy labeling and high stability against hydroly-sis and heat treatment [9,10]. These properties enabledthe use of the HRP-DNAzyme as a versatile label for nu-merous sensors including enzyme [11], DNA [12], apta-mer complexes [13], cancer biomarker [14, 15] and metalion [16] sensors. It is probably the most frequently usedbiocatalytic DNAzyme label for amplified biosensing andalso has been used as electrocatalyst for amplified sensing[17].

As is well-known, amplification is an important aspectin bioanalytical science [18], so the amplified detectionhas attracted substantial research efforts in the past fewyears. For instance, numerous electrical [19,20], optical[21–23] and microgravimetric [24,25] amplified sensorshave been developed rapidly. Among them, electrochemi-cal amplified methods are powerful analytical techniquesfor moderate cost, instrumental simplicity, high sensitivityand high compatibility [26,27]. Different electrochemicalstrategies have been explored to achieve signal amplifica-tion, including the use of nanoparticles, conducting poly-mers and catalysis [28–31]. Therefore, electrocatalysisrepresents a preferred means of signal amplification [28–31], which avoids some complicated synthesis processesand introduces other interfering agents.

Methylene blue (MB), a water-soluble polynuclear aro-matic dye (typical phenothiazine dye), which exhibitsa high electrocatalytic activity on various electrode surfa-ces, is known to act as a redox reporter or electron-trans-fer mediator for some redox enzymes [28–32]. It is alsoreported that enzymes such as horseradish peroxide andglucose oxidase have been shown to readily interact elec-tronically with phenothiazine dyes MB [33–38]. In addi-tion, MB is typically used as the redox reporter for DNA-mediated electrocatalysis [39]. Inspired by the interaction

Abstract : Electrocatalysis of redox enzymes shows wideapplication for biosensing. DNAzymes exhibiting specificcatalytic activities have aroused great interest recently.However, there are few studies on the electrocatalysis be-tween DNAzyme and electron mediator. In this paper,based on the electrocatalysis of methylene blue (MB) andhorseradish peroxidase mimicking DNAzyme (HRP-DNAzyme), an amplified electrochemical biosensor forthe detection of adenosine triphosphate (ATP) was de-signed. In the present system, by means of the ATP-apta-mer interaction, two guanine-rich DNA sequences, one ofwhich was labeled with MB at the 5’ end, were assembled

on the gold electrode. In the presence of K+ and hemin,the guanine-rich DNA sequences transferred to HRP-DNAzyme. The conformational change of the structureresulted in the approaching of MB and HRP-DNAzymewhich made the electrocatalytic process between MB andHRP-DNAzyme possible. We used cyclic voltammetryand electrochemical impedance spectroscopy to study theelectrocatalytic process. The system was therefore utilizedfor amplified detection of ATP without imposing any newconstraints to the platform which showed satisfactoryresult.

Keywords: Aptasensor · Electrocatalysis · Horseradish peroxidase · DNAzyme · G-quadruplex

[a] G. Wang, L. Chen, Y. Zhu, L. Wang, X. ZhangCollege of Chemistry and Materials Science, Anhui KeyLaboratory of Chem-Biosensing, Anhui Key Laboratory ofFunctional Molecular Solids, Anhui Normal University,Wuhu 241000, P R Chinafax: +86-553-3869303; tel: +86-553-3869303*e-mail: [email protected]

[email protected]

www.electroanalysis.wiley-vch.de � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2014, 26, 312 – 318 312

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of MB with redox enzymes and the peroxidase-like activi-ty of horseradish mimicking peroxidase, it is hopeful touse the horseradish peroxidase mimicking DNAzyme aselectron sinks and MB as redox reporter for electrocatal-ysis in DNA sensor.

In this paper, based on the redox reporter role of MB,the character of HRP mimicking DNAzyme and their in-teraction, new paradigms of electrocatalysis amplificationhave been developed with MB and horseradish perox-idase mimicking DNAzyme. For sensing amplificationassay, adenosine triphosphate (ATP), a multifunctionalnucleotide in living organisms, which plays a critical roleas an energy currency of the cell in cell physiology andmakes a difference in indicating the cell viability and cellinjury, was chosen as a model in this system [40,41]. Theelectrocatalysis of MB with horseradish peroxidase mim-icking DNAzyme can be utilized for ATP amplified de-tection strategies without imposing any new constraints tothe platform, with a low detection of 10 nM and widelinear range of 10�8–10�3 M.

2 Experimental

2.1 Chemicals and Materials

HPLC-purified oligonucleotidesS1: 5’-SH-(CH2)6-GTATATTTGGGATGGGTTT-

CTGGGGG-AGTA-3’;S2: 5’-MB-ATT-TGCGGGAGGAAGTTTGGGTAG-

GGT-3’;S3: 5’-ATTTGCGGGAG-GAAGTTTGGGTAGGGT-3’,Tris (hydroxymethyl) aminomethane (Tris), ATP and

hemin were obtained from Sangon Biotechnology Co.Ltd. (Shanghai, China). All other reagents used in this ex-periment were obtained from the Sinopharm ChemicalReagent Co. Ltd. (Shanghai, China). These reagents weredissolved in ultrapure water and ultrapure water was usedthroughout the experiments.

2.2 Apparatus

Electrochemical measurements were carried out usinga CHI 660C electrochemistry workstation (Shanghai CHInstruments Co., China) at ambient temperature (22�2 8C). A three-compartment electrochemical cell con-tained a modified gold electrode (GE, d=2 mm) as work-ing electrode, a platinum wire as auxiliary electrode anda saturated calomel electrode (SCE) as reference elec-trode. All potentials were measured and reported versusthe SCE in this experiment. All the electrochemical ex-periments were carried out at room temperature. Centri-fugation was performed using a HERMLEZ 36 HK appa-ratus (Wehingen, Germany). Solutions were prepared byusing ultrapure water processed by PSDK2-10-C (Beijing,China). The pH values of the phosphate saline buffer(PBS) and Tris-HCl were measured with a glass electrodeconnected to a PHS-3C pH meter (Shanghai, China).

2.3 The Preparation of MB/HRP-DNAzyme ModifiedGold Electrode (MB/HRP-DNAzyme/GE)

Before use, these oligonucleotides were dissolved in0.05 M Tris-HCl buffer (pH 7.4) and stored at 4 8C forone night. When used in the experiment, the oligonucleo-tides solutions were first heated to 80 8C and slowlycooled to room temperature. The gold electrodes iscleaned as following, polishing the gold electrodes on mi-crocloth with 0.3 and 0.05 mm alumina powder, sonicatingthem in ethanol and ultrapure water for 5 min, respective-ly, and lastly removing any remaining impurities via elec-trochemical cleaning, scanning the electrode in 0.1 MH2SO4 between �0.2 and 1.55 V at 100 mV/s untila steady-state redox wave observed. At this moment, theimmobilization of the capture probe (S1) was performedby dropping 30 mL of 10 mM Tris�HCl buffer (pH 7.4)containing 1.0 mM S1 probe and 1.0 M NaCl on thecleaned gold electrodes (bare GE) in a humidified cham-ber for 2 hours. The self-assembly was proceeded throughthe covalent bond with thiol of S1 and Au atom. Afterbeing thoroughly rinsed with the washing buffer (10 mMTris-HCl, pH 7.4, 100 mM NaCl), the S1 probe modifiedgold electrode (S1/GE) was incubated in the hybridiza-tion buffer 0.05 M Tris-HCl buffer (pH 7.4) involving1 mM S2 containing the label of MB, 1 mM ATP for 2hours (S1/ATP/S2/GE). Then, ATP-aptamer complexmodified electrode was incubated in the solution contain-ing 100 mM KCl, 400 mM NaCl and 1 mM hemin for2 hours to form the G-quadruplex structures on the elec-trodes named MB/HRP-DNAzyme/GE. For the detectionof ATP, the sensor was prepared under the same condi-tions except incubated with different concentrations ofATP. For the control experiment, S1 modified electrodeincubated only with S2 (without ATP) was prepared andnamed as S1/S2/GE. Then it was also incubated with1 mM hemin and 100 mM K+ (S1/S2/GE-hemin/K+). TheS1 modified electrode was incubated with ATP and S3,which has the same sequence of S2 but has not beenmodified with the redox reporter MB, was named S1/ATP/S3/GE. After it was incubated with 1 mM hemin and100 mM K+, HRP-DNAzyme/GE was formed withoutMB modified on it.

2.4 Measurement Procedure

Cyclic voltammetry (CV) was performed in 0.05 M Tris-HCl buffer (pH 7.4) within a potential range from 0 to�0.5 V using a scan rate of 50 mV/s. Electrochemical im-pedance spectroscopy (EIS) measurements were carriedout in the presence of a 5 mM K3[Fe(CN)6]/K4[Fe(CN)6](1 :1) mixture as redox probe in 0.1 M PBS (pH 7.4) con-taining 0.1 M KCl at a bias potential of 0.18 V. The alter-native voltage was 5 mV and the frequency range was 0.1KHz–10 KHz.


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3 Results and Discussion

3.1 The Design of the Sensor

The schematic diagram for the detection of ATP by elec-trocatalysis with DNAzyme and MB is shown inScheme 1. First, the thiolated nucleic acid sequence (S1),containing one part of aptamer sequence for ATP (itali-cized in S1 of Section 2.1) and guanine-base-rich se-quence (underlined in S1 of Section 2.1), was self-assem-bled on the gold electrode surface by Au-S covalentbond. Subsequently, the S1 modified electrode incubatedwith ATP and another nucleic acid sequence with MB la-beled at its 5’end, S2, which contained another part of theaptamer of ATP (italicized in S2 of Section 2.1) and alsoa guanine-base-rich sequence (underlined in S2 of Sec-tion 2.1). By the reaction of ATP and its two parts of ap-tamers, ATP-aptamer complex modified electrode wasformed with two guanine-base-rich sequences hanging.Then in the presence of K+ and hemin, the two guanine-base-rich sequences folded into G-quadruplex to formHRP-DNAzyme. Because MB molecule was labeled inS2, now the folding of S1 and S2 made MB and HRP-DNAzyme approaching near the electrode surface. WithMB as the redox reporter and HRP-DNAzyme as theelectron sink near the surface of the electrode, electroca-talysis may be realized as expected. The electrocatalyticsignal is related with the concentration of ATP which be-comes the base of its quantitative analysis.

3.2 EIS Characterization of the Electrode ModifyingProcess

The EIS characterization of the electrode modifying pro-cess is shown in Figure 1. The step-wise modification pro-cess of the gold electrode is obtained in the form of a Ny-quist plot. Ret, the surface electron transfer resistance ofthe electrode, indicates the surface electron transfer. As

we can see, a very small semicircle domain (curve a) wasobserved on the bare gold electrode illustrating that thebare gold electrode displays a very fast electron transferprocess. When the gold electrode is modified with S1, theRet gives an obvious rise (curve b) indicating that the cap-ture sequence can severely hinder the diffusion of ferri-cyanide toward the electrode surface. After incubatingthe S1/GE with S2 and ATP, the Ret gives a successiverise as curve c shows, which is attributed to the comple-mentary pairing of ATP and its aptamer that retards theelectron transfer tunnel. After that, with the ATP-apta-mer complex modified electrode incubated with K+ andhemin, the value of Ret increases again (curve d). It maybe because that the formation of G-quadruplex and theexistence of hemin increase the steric hindrance of theelectron transfer. This shows that the process may be inaccordance with what we described before.

Scheme 1. The fabrication process of the aptasensor.

Fig. 1. EIS of the electrodes in different procedures: bare GE(curve a); S1/GE (curve b); S1/ATP/S2/GE (curve c); MB/HRP-DNAzyme/GE (curve d).


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3.3 The Performance of the Modified Electrode

Cyclic voltammetry was employed to validate the fabrica-tion of the sensing interface as Figure 2A shows. Curvesa and b show both bare and S1 modified gold electrodeshave low electrochemical responses in the blank buffersolution. However, the S1 modified electrode incubatedin the solutions containing S2 (curve c) or S2 with ATP(curve d) also shows no obvious change from CV re-sponse, respectively. That means MB alone has not a cata-lytic effect in the system. Then as curve e shows, the elec-trochemical response has an apparent enlargement afterincubating the S1/ATP/S2/GE with K+ and hemin, whichmay be due to that electrocatalysis may be achieved be-tween the formed HRP-DNAzyme structure and MB[39]. In order to further validate the procedure of theelectrochemical amplification, control experiments wereperformed. Figure 2B shows an obviously lower response,when S1/S2/GE was incubated in solutions containinghemin and K+ without ATP (curve a of Figure 2B), thanthat of curve b of Figure 2B in which S1/S2/GE was incu-bated in solutions containing hemin, K+ and ATP. Wesuggest that S2 can not be conjugated with S1 in the ab-sence of ATP and no electrocatalysis is realized then. Inorder to test the role of MB, S3 containing the same se-quence as S2 but without MB label was designed. Curvea in Figure 2C is the CV response on the HRP-DNAzymealone (without MB) modified electrode (HRP-DNA-zyme/GE), which was formed by S1/ATP/S3/GE incubat-ed with hemin and K+ . As Figure 2 C shows, MB/HRP-DNAzyme/GE shows obvious CV response (curve b) andscarcely any magnification of electrochemical responseemerges on the HRP-DNAzyme/GE (curve a), whichproves that MB plays an important role in the electroca-talytic signal amplification. According to the literature[39] and the result of Figure 2, we can suggest that thegeneral electrocatalytic process developed in our systemwith MB and HRP-DNAzyme is carried out by the fol-lowing processes: first, MB was reduced to leucomethy-lene blue (LB) with two electrons and then with twoequivalents of freely diffusing electrons, LB was proceed-ed to electrochemically interact with, and further reduceDNAzyme, returning LB to its oxidized form MB andcompleting the electrocatalytic process. The redox processis summarized by equations as follows:

MBþ 2e! LB

LBþHRP-DNAzyme ðOxÞ !MBþHRP-DNAzyme ðRedÞ

3.4 Optimization of the Variables of the System

In this experiment, experimental conditions were opti-mized to achieve high sensitivity. Figure 3 shows the opti-mization of the variables of the system, such as pH value,reaction temperature and incubation time. The effect ofpH on the system in the first incubation step of the apta-mer-ATP interaction was studied over the pH range 5.0–

10.0. As shown in Figure 3A, the current enlarges with in-creasing pH value in the range from 5.0 to 7.5. However,when the pH is higher than 7.5, the response decreases

Fig. 2. Cyclic voltammetry of different electrodes in Tris-HCl(pH7.4): (A) bare GE (curve a); S1/GE (curve b); S1/S2/GE(curve c); S1/ATP/S2/GE (curve d); MB/HRP-DNAzyme/GE(curve e); (B) S1/S2/GE-hemin/K+ (curve a); MB/HRP-DNA-zyme/GE (curve b); (C) HRP-DNAzyme/GE (curve a); MB/HRP-DNAzyme/GE (curve b).


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gradually. Figure 3B depicts the effect of reaction temper-ature ranging from 25 to 40 8C for the incubation of theaptamer-ATP. It is obvious that the response increaseswith the temperature rising from 25 to 34 8C and then de-creases rapidly as the reaction temperature increasesfrom 34 to 40 8C. The different incubation time periods ofthe aptamer-ATP interaction on the signal response arestudied from 20 to 140 min as shown in Figure 3C. The re-sponse increases with the time lasting from 20 to 120 min

and then decreases rapidly from 120 to 140 min. Thus theoptimized reaction conditions are 35 8C under pH 7.4 with120 min for incubation of the aptamer-ATP throughoutthe experiments.

3.5 Determination of the ATP with the MB/HRP-DNAzyme System

Under the optimized conditions, different concentrationsof ATP were further tested in the MB/HRP-DNAzymesystem. The CV responses of the present MB/HRP-DNA-zyme system toward different concentrations of ATP areshown in Figure 4A. The electrocatalytic currents enlargealong with the concentration of ATP increasing. Fig-ure 4A Inset shows the linear relationship from the oxida-tion response of the sensor with the logarithm of ATPconcentration between 10�8 and 10�3 M with a good cor-relation coefficient 0.9942 and the detection limit is10 nM (3 n/s, where n was the standard deviation of theintercept and s was the slope of the calibration curve). It

Fig. 3. Optimization of experimental conditions: (A) pH value;(B) incubation temperature; (C) hybridization time.

Fig. 4. (A) MB/HRP-DNAzyme/GE incubated with differentconcentrations of ATP, (a–f): 10�8, 10�7, 10�6, 10�5, 10�4, 10�3 M.Inset: Corresponding calibration curves of current vs. the loga-rithm of the ATP concentration. (B) Selectivity in the analysis ofATP by the MB/HRP-DNAzyme system. The concentration ofeach molecule was 1 mM.


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is suggested that the high sensitivity dates from the am-plification path afforded by the electrocatalytic system.Because its simple hybridization of two oligonucleotideprobes in one step, while other recently studied ATP sen-sors usually included multistep hybridization of three oli-gonucleotide probes, the present electrochemical ATP de-tection system also has the advantages of simplicity indesign and easy operation over other recently developedelectrochemical ATP sensors. (Table 1).

3.6 The Specificity, Stability and Reproducibility of theSensor

In order to investigate the selectivity of the MB/HRP-DNAzyme system, we study the interference of ATP onthe present sensor. As shown in Figure 4B, the specificityof the method for ATP was tested by substituting ATPwith kinds of its molecular analogs such as guanosine tri-phosphate (GTP), cytidine triphosphate (CTP) and uri-dine triphosphate (UTP) at 1 mM concentration, respec-tively. As we can see, the presence of interfering mole-cules exhibits no significant effect on the electrochemicalintensity and only in the absence of ATP, it emerges obvi-ous signals, revealing the high selectivity of the proposedapproach. Therefore, the proposed aptasensor presentsremarkably high sensitivity and selectivity. Such excellentselectivity should be ascribed to the specific coordinationin the hybridization of the signal probe to the captureprobe with the aptamer. On the other hand, the stabilityand reproducibility of the sensor are also examined re-spectively. The stability of the sensor was examined bymonitoring the current response after successive cyclingthe modified electrode in the potential range from 0 to�0.5 V in 0.1 M Tris-HCl for 500 circles. It is found thatthe current retains 89.3 % of its initial value and no obvi-ous potential shift is observed. It is found that the currentresponse does not apparently change in the first succes-sive eight days by everyday use. Only 15.1% leakage isfound after 3 months. Thus, it shows that the sensor haslong-term stability and reproducibility.

4 Conclusions

In this paper, we have introduced a facile, inexpensive,sensitive and selective electrochemical assay for the de-tection of the ATP based on the MB/HRP-DNAzyme

system. In such an approach, amplification of the signal isachieved significantly by the electrocatalysis of MB withhorseradish peroxidase mimicking DNAzyme. Thus, bymeans of the electronic interaction between this mimick-ing DNAzyme and MB, electrochemical detection of theATP was enforced. This assay provides a good linear rela-tionship with the concentration of ATP in a wide rangeand a detection limit of 10 nM. No specialized backfillingagents or assembly conditions were required, demonstrat-ing the general applicability of this system. Most impor-tantly, the enhanced sensitivity permits nanomole ATPsampling. In addition, other related molecules can be de-tected in the similar way just with the need to design a dif-ferent DNA sequence.

Acknowledgements

This work was financially supported by the Projects(21073001, 21005001 and 21371007) from the NationalNatural Science Foundation of China, the Natural ScienceFoundation of Anhui (KJ2009B013Z), the Project ofAnhui Key Laboratory of Controllable Chemistry Reac-tion & Material Chemical Engineering (OFCC0905).

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Method Linear range Detection limit References

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Received: September 3, 2013Accepted: October 13, 2013

Published online: November 26, 2013


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Adenosine Triphosphate Sensing by Electrocatalysis with DNAzyme