A Strategy for Development of Electrochemical DNA Bio Sensors

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Accepted Manuscript

Title: A Strategy for Development of Electrochemical DNABiosensor based on Site-specific DNA Cleavage of Restriction

Endonuclease

Authors: Jinghua Chen, Jing zhang, Huanghao Yang, Fengfu

Fu, Guonan Chen

PII: S0956-5663(10)00301-5

DOI: doi:10.1016/j.bios.2010.05.033

Reference: BIOS 3807

To appear in: Biosensors and Bioelectronics

Received date: 12-3-2010

Revised date: 7-5-2010

Accepted date: 24-5-2010

Please cite this article as: Chen, J., zhang, J., Yang, H., Fu, F., Chen, G., A

Strategy for Development of Electrochemical DNA Biosensor based on Site-specific

DNA Cleavage of Restriction Endonuclease, Biosensors and Bioelectronics (2008),

doi:10.1016/j.bios.2010.05.033

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A Strategy for Development of Electrochemical DNA1

Biosensor based on Site-specific DNA Cleavage of 2

Restriction Endonuclease3

4

Jinghua Chena,b

, Jing zhangc, Huanghao Yang

a*, Fengfu Fu

a, Guonan Chen

a*5

a. Ministry of Education Key Laboratory of Analysis and Detection Technology for 6

Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection7

Technology for Food Safety, Department of Chemistry, Fuzhou University,8

Fuzhou, 350002, China9

b. Department of Pharmaceutical Analysis, Faculty of Pharmacy, Fujian Medical10

University, Fuzhou 350004, China.11

c. Pharmaceutical department of Fujian College of Medical Occupation and12

Technology, Fuzhou 350101, China13

14

Abstract15

A new strategy for development of electrochemical DNA biosensor based on16

site-specific DNA cleavage of restriction endonuclease and using quantum dots as17

reporter was reported in this paper. The biosenser was fabricated by immobilizing a18

capture hairpin probe, thiolated single strand DNA labeled with biotin group, on a gold19

electrode. BfuCI nuclease, which is able to specifically cleave only double strand DNA20

but not single strand DNA, was used to reduce background current and improve the21

sensitivity. We demonstrated that the capture hairpin probe can be cleaved by BfuCI22

* Cooresponding author, e-mail: [email protected] (G. Chen); [email protected] (H. Yang); Tel.: +86 591

87893315; fax: +86 591 83713866



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nuclease in the absence of target DNA, but can not be cleaved in the presence of target23

DNA. The difference before and after enzymatic cleavage was then monitored by24

electrochemical method after the quantum dots were dissolved from the hybrids. Our 25

resuts suggested that the usage of BfuCI nuclease obviously improved the sensitivity26

and selectivity of the biosensor. We successfully applied this method to the27

sequence-selective discrimination between perfectly matched and mismatched target28

DNA including a single-base mismatched target DNA, and detected as low as 3.3×10-14

29

M of complementary target DNA. Furthermore, our above strategy was also verified30

with fluorescent method by designing a fluorescent molecular beacon (MB), which31

combined the capture hairpin probe and a pair of fluorophore (TAMRA) and quencher 32

(DABCYL). The fluorescent results is consistent with that of electroanalysis, further 33

indicated that the proposed new strategy indeed works as our expected.34

Keywords: Electrochemical DNA Biosensor, Site-specific DNA Cleavage, Restriction35

Endonuclease, Cymbidium mosaic virus36

37

1. Introduction38

Methods for the sequence-specific DNA detection have attracted significant39

attention due to possible applications in fields ranging from virus detection to the40

diagnosis of genetic diseases [Heller et al., 2002; Balakin et al., 1998]. Consequently,41

various techniques have been employed for the detection of DNA hybridization, such42

as chromosome analysis [Jorge et al., 1996], fluorescence in situ hybridization (Jilani43

et al., 2008) and real-time quantitative reverse transcription PCR (Rong et al., 2002).44



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But there were some limitations in these techniques, such as time-consuming, poor 45

precision and expensiveness. So it is very significant to develop new effective46

methods.47

Within recent years, several inventive designs for DNA sensors based on an48

electrochemical readout have appeared due to the fact that electrochemical detectors49

are simple, reliable, cheap, sensitive and selective for genetic detection [Miao et al.,50

2003, 2004]. These sensors can be prepared by immobilizing single-stranded DNA51

probes on different electrodes and using electroactive indicators or other methods to52

measure the hybridization events between the DNA probes and their complementary53

DNA fragments. Consequently, a variety of sensing strategies have been developed,54

aiming at the improvement of sensitivity and selectivity. For example, DNA55

hybridization detection deals with the use of electroactive indicators interacting56

directly and specifically with the DNA duplex, such as intercalators, DNA groove57

binders, metal complexes and threading agents, have been recently studied58

(Demeunynck et al., 2003; Li et al., 2007; Liu et al., 2004; Niu et al., 2006; Chen et al.,59

2003, 2004). While intercalator-based DNA sensing protocols take the advantages of 60

design simplicity and operation convenience, they often suffer from high background61

signals that are associated with non-specific binding of intercalators to unhybridized62

ssDNA.63

In an attempt to circumvent this problem, DNA hairpins were proposed and64

popularly employed. DNA hairpins have been found to exhibit extraordinary stability,65

better selectivity and higher specificity than similar assays performed using66



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single-stranded DNA probe. Furthermore, DNA probes with hairpin structure exhibit a67

particularly high sensitivity to detect mismatch. Consequently, their hybridization68

processes can significantly enhance the specificity and improve the signal-to-noise69

ratio, thus enabling the detection of as few as picomolar DNA targets. The detection70

limit can be further pushed down to the femtomolar level by coupling the hairpin-type71

sensors with signal amplification offered by using either enzymes (Liu et al., 2008) or 72

inorganic nanoparticles labels (Gao et al., 2006).These strategies greatly improved the73

sensitivity, however, practical application of these strategies might be hampered due to74

poor stability of enzymes or relatively poor specificity for the detection of single base75

mismatch and single-nucleotide polymorphisms (SNPs). Moreover, it is important to76

develop a simple and rapid approach to realize simultaneous detection of multiple77

targets. Existing sensors are often not amenable to detection in complicated samples78

such as DNA species related to virus. So, new sensitive and selective DNA79

hairpin-type strategies still need to be developed.80

Cymbidium mosaic virus (CymMV) is one of the most prevalent and economically81

important orchid viruses (Wong et al., 1994). It infects numerous commercially82

important orchid genera and has attained a worldwide distribution (Zettler et al., 1990).83

CymMV cause flower colour breaking, size reduction and stunted growth, thus84

reducing the quality of the orchids and greatly affecting the economy of the orchid85

industry. Therefore, it is very significant to develop a new effective method for 86

detection of CymMV. Herein, we reported a new strategy for development of 87

electrochemical DNA biosensor for detection of DNA species related to virus88



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(cymbidium mosaic virus, CymMV, in this case) using site-specific DNA cleavage of 89

restriction endonuclease (REN)–BfuCI. A restriction enzyme (or restriction90

endonuclease) is an enzyme that cuts double-stranded or single stranded DNA at91

specific recognition nucleotide sequences known as restriction sites (Kessler and92

Manta,1990). In this case, BfuCI is able to specifically cleave only double strand DNA,93

but not single strand DNA. It have been widely used in life processes, such as the94

replication, transcription, recombination and repair of nucleic acids, however using95

BfuCI in electrochemical biosensors have not been reported. In our strategy, the96

capture probe, thiolated hairpin DNA sequence labeled with biotin group at the other 97

end, was immobilized on a gold electrode through S-Au bonding (Figure 1). The stem98

of hairpin DNA is a special double-stranded DNA that contains a BfuCI REN99

recognition site (-mer sequence: 5′-GATC-3′) (Shizuka et al., 2008).The electrode was100

then blocked with 2-mercaptoethanol (MCH) to form a mixed monolayer. MCH has101

been used as a spacer to minimize nonspecific binding and maximize the efficiency of 102

hybridization of capture and target probes (Cheng et al., 2007). Next, the immobilized103

biotin hairpin probes were hybridized with various target DNA sequences, then the104

hybridized electrode surface was incubated with REN to discriminate between105

perfectly matched target DNA (PM, complementarity with the loop part of hairpin106

probe) and mismatched target DNA (MM), including a single-base mismatched (SM)107

target DNA.108

109

(Figure 1 should be inserted in here)110



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111

2. Experimental112

2.1. Chemicals and Apparatus113

All oligonucleotides were synthesized by TaKaRa biotechnology Co., Ltd. (Dalian,114

China), and their base sequences were illustrated in Table 1. BfuCI buffer were115

obtained from BoCai Biotechnology Co. Ltd. (Shanghai, China). Avidin-QDs (with a116

CdSe/ZnS core-shell structure about 10 nm) were obtained from Wuhan Jiayuan117

Quantum Dots Co., Ltd. (Wuhan, China). Tris-(hydroxymethyl) aminomethane was118

purchased from Cxbio Biotechnology Co. Ltd. (Denmark). Ethylenediaminetetraacetic119

acid (EDTA), mercaptohexanol (MCH) and tris (2-carboxyethyl) phosphine120

hydrochloride (TCEP) were purchased from Sigma-Aldrich (USA). The buffer 121

solutions are as follows: Hybridization buffer was the mixture of 100 mM NaCl and 10122

mM TE (pH 8.0), buffers for DNA immobilization buffer is the mixture of 10 mM TE,123

10 mM TCEP, 100 mM NaCl and 10 mM MgCl2 (pH 7.4), MgCl2 was added into the124

electrolyte to induce the formation of the hairpin structure of cDNA assembled onto125

the electrode surface, as reported previously (Dai et al., 1998; Dao et al., 1992; Gao et126

al., 1999; Ramsing et al., 1989). Washing buffer was the mixture of 0.1 M NaCl and 10127

mM PB (pH 7.4). Solution contained 0.1 mol/L KCl and 2 mmol/L128

Fe(CN)63-

/Fe(CN)64-

was used for electrochemical impedance spectroscopy (EIS)129

characterization. All solutions were prepared with MilliQ water (18.2 MΩ/cm130

resistivity) from a Millipore system.131

The electrochemical measurements for electrochemical impedance spectroscopy132



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(EIS), stripping voltammetry and differential pulse voltammetry (DPV) were carried133

out on a CHI 660C electrochemical working station (CH Instrument Company, USA)134

using a three-electrode system consisted of a platinum wire as an auxiliary electrode,135

an Ag/AgCl electrode as reference electrode and a 2-mm-diameter Au disk electrode as136

working electrode (for EIS) or a glassy carbon electrode as working electrode (for 137

stripping voltammetry and DPV). The spectra and intensity of fluorescence were138

measured with a Eclipse spectrofluorometer (Varian).139

2.2. Electrode Preparation140

The whole fabrication process of this biosensor is outlined in Figure 1. A gold disk 141

electrode (GE) was firstly polished to obtain mirror surface with 0.05 μm alumina142

powder, followed by sonication in ethanol and water for 5 min respectively. Then, the143

GE was electrochemically cleaned to remove any remaining impurities (Fan et al.,144

2003). After drying with nitrogen, the electrode was immediately used for DNA145

immobilization. Firstly, 5 µL S1 solution was first spread on the pre-cleaned gold146

electrode surface for 12 hours in the 100 % humidity. Next, this electrode was147

immersed in 1 mmol/L MCH for 2 hour to remove the nonspecific DNA adsorption148

and optimize the orientation of the capture probes to make hybridization easier (Zhang149

et al., 2006). Then, the S1-immobilized electrode was immersed in hybridization buffer 150

containing complementary T1, single-base mismatch T2 or uncomplementary T3,151

respectively. The hybridization was allowed for 30 min with stirring at 45

o

C.152

Afterwards, the sensor was then immersed in a solution of BfuCI reaction buffer 153

(0.067 units/uL) in NEBuffer 41×(50 mM potassium acetate, 20 mM Tris-aceate, 10154

mM magnesium acetate, 1 mM dithiothreitol, pH 7.9) and rinsed with wash buffer.155

Prior to electrochemical measurement, the modified electrode was incubated with 3 µL156



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of Avidin-QDs for 25 min at room temperature and washed with wash buffer to remove157

the physical adsorption of QDs and dried under a stream of nitrogen.158

2.3. Electrochemical Detection159

The Au electrode modified with hybrids labeled with biotin-avidin-system was160

immersed into a colorimetric tube containing 200 μL of 0.2 M nitric acid solution for 5161

min. The dissolved Cd2+

solution was transferred into 2.0 mL of the 0.1 M pH 5.3162

HOAC-NaAC buffer supporting electrolyte solution containing 10 μg/mL HgCl2.163

Stripping voltammetric measurements of the dissolved Cd2+

were performed using an164

in situ plated mercury film on a glassy carbon electrode following a pretreatment at 0.6165

V for 1 min, and a accumulation at -1.4 V for 5 min. The positive (DPV) scan was166

performed after a 15 s rest period from -0.8 to -0.5V (vs. Ag/AgCl), with pulse167

amplitude of 50 mV and pulse width of 50 ms. The anodic stripping peak current i p, a168

located at about -0.68 V was taken as the analytical response.169

Electrochemical impedance experiments were performed in the presence of 2 mM170

[Fe(CN)6]4-/3-

. The biased potential was 0.22 V (versus Ag/AgCl) and the amplitude171

was 5.0 mV. The electrochemical impedance spectra (EIS) were recorded in the172

frequency range of 0.1-105

Hz with a sampling rate of 12 points per decade. A Nyquist173

plot (Zre vs Zim) was drawn to analyze the impedance results.174

2.4. Investigation of specificity by fluorescence175

Under the optimum condition, the molecular beacons were added in NEBuffer 41×176

(50 mM potassium acetate, 20 mM Tris-aceate, 10 mM magnesium acetate, 1 mM177

dithiothreitol, pH 7.9) containing BfuCI (0.067 units/uL), complementary DNA or 178



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complementary DNA+BfuCI (0.067 units/uL), respectively. The hybridization was179

allowed for 30 min with stirring at 45oC. Afterwards, in the determined excitation and180

emission wavelengths (λ ex=521 nm, λ em=586 nm), the fluorescence intensity of 181

molecular beacons and the fluorescence intensity hybridization buffer were detected182

individually. Compare the change of the fluorescence intensity and investigate the183

specificity.184

3. Results and discussion185

3.1. Strategy for design of this Electrochemical DNA Biosensor186

In the absence of PM target, the capture probe exits predominantly in the hairpin187

form (loop-stem structure, at certain concentrations and buffer conditions). So it can be188

easily cleaved by REN. Thus, avidin labeled QDs (Avidin-QDs) cannot bind to the189

capture probe through the biotin-avidin-system. However, in the presence of the PM190

target, the loop part of the hairpin probe was hybridized with the target strand to form191

the matched duplex DNA. Meanwhile, the stem is opened to form single strand and192

resistant to the cleavage of REN. Thus the Avidin-QDs can then bind to the capture193

probe. So, the hybridization events can be monitored by stripping voltammetry after 194

the QDs were dissolved from the hybrids. In contrast to prior electrochemical DNA195

detection schemes based on DNA hairpins (Immoos et al., 2004; Fan et al., 2003), this196

strategy generates an electrochemical signal upon recognition of the target DNA (i.e.,197

signal “on” device).198

3.2 EIS of different modified electrodes199

Electrochemical impedance technique was employed to characterize the fabrication200



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in whole process. Figure 2 shows the EIS changes for surface-modified process.201

Compared with the bare Au electrode (Figure 2a), the probe S1 modified and MCH202

treated Au electrode (S1/MCH/GE) shows a larger eT resistance (Figure 2b), mainly203

due to the electrostatic repulsion between negative charges of the DNA backbone and204

the Fe(CN)63−/4−

probe. After the probe modified electrode was immersed into the205

BfuCI reaction buffer solution, the eT resistance (Figure 2c) decreased. The resistance206

decrease could be attributed to the fact that the stem of the hairpin probe structure207

contains a cleavage site for BfuCI and was subsequently cleaved by this enzyme. The208

cleavage resulted in the decrease of the negative charges of DNA on the GE surface.209

Under optimal condition, the S1 can hybridize with target DNA T1 and become210

double-stranded DNA (S1-T1/MCH/GE). At this time, the monolayer of DNA on the211

electrode surface became denser, and the negative charges on the electrode surface212

increased. Thus the electron-transfer resistance is enhanced further, and the value of 213

Ret increases (Figure 2d). When the S1-T1/ MCH/GE was directly combined with214

BfuCI, no significant difference of eT resistance was observed (Figure 2e). It may be215

that the formation of S1-T1 structure essentially does not contain the REN cleave site.216

Therefore, it can not be cleaved by BfuCI, which indicated that the hybridization217

events could be monitored by employing this novel electrochemical strategy.218

219


221

3.3. Sensitivity of the DNA Biosensor222



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The sensitivity of the DNA biosensor was detected as shown in Figure 3. The results223

showed that the peak current value of DPV increased with the concentration of the224

target DNA. The linear range for logarithm of target DNA was 1.0×10-13

~1.8×10-12

M225

with the equation of I(μA)=0.1366logC(0.1 pM) + 0.1953 (I was the current intensity,226

and C was the concentration of target DNA, r=0.9956, n=6). The relative standard227

deviations (RSD) of the sensor for 10 replicate determination of 1.0×10−12

M target228

DNA was 2.8 %. The detection limit of 3.3×10-14

M target DNA could be estimated229

using 3σ. This detection limit is lower than some existing electrochemical biosensors230

based on quantum dots as reporter (Sun, et al., 2008; Wang et al., 2003; Zhu et al.,231

2004).232

233

234


236

3.4. Selectivity of the DNA Biosensor237

The selectivity of the present biosensor was investigated by using the DNA probe238

(S1) to hybridize with the same concentration of complete complementary target DNA239

sequence (T1), the single-base mismatched DNA sequence (T2) and the240

noncomplementary DNA sequence (T3), respectively. As shown in Figure 4, a241

well-defined peak current signal of Cd2+

was obtained for the complementary sequence242

(Figure 4, curve a). It can be also known that the present biosensor has high243

hybridization specificity, it can easily discriminate the complementary from244



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single-base mismatch target DNA. In the presence of oligonucleotide containing a245

single-base mismatch, significantly decreased voltammetric signal can be observed246

(Figure 4, curve b), which indicates that the complete hybridization is not247

accomplished due to the base mismatch. In addition, as expected, nearly no response of 248

peak current can be observed for the capture probe hybridization with249

noncomplementary sequence oligonucleotide (Figure 4, curve c), since no successful250

hybridization occurs due to the sequence mismatch between the capture probe and the251

noncomplementary sequence.252

253


255

The selectivity and specificity of the hairpin DNA probe was also studied by using256

fluorescence method, which was valuable in optimization of the hairpin DNA probe257

design. The fluorescent molecular beacons (MB) was designed by a combination of 258

DNA hairpin structure and a pair of fluorophore (TAMRA) and quencher (DABCYL).259

The ends of the stem of the hairpin were modified by fluorophore and quencher,260

individually. Before hybridization, the structure of DNA probe kept hairpin, and the261

quencher was close to the fluorophore. At this time, the efficiency of fluorescence262

quenching played a key role. So there were little fluorescent signals on the DNA probe263

(Figure 5, curve 1). After hybridization with the complementary DNA, with the264

hybridization of DNA and the change of the hairpin structure, the distance between the265

fluorophore and the quencher was increased. So the efficiency of fluorescence266



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quenching between the fluorophore and the quencher was decreased. Thus the267

fluorescence intensities were increased. (Figure 5, curve 3). It is worth mentioning that268

the fluorescence intensities were significantly increased after MB was directly269

incubated with BfuCI nuclease (Figure 5, curve 2). It could be attributed to the fact that270

when the MB is incubated with solutions containing BfuCI, the BfuCI carries out271

catalytic reactions to cleavage of the MB at the cleave site. Then the strand is broken272

into two pieces and dissociated from the MB. As a result, the amount of the quencher 273

decreases, resulting in higher fluorescence intensities. However, after hybridization274

with the PM target DNA, the stem of the MB was opened, meanwhile, the new double275

helix structure of the DNA system does not contain the REN cleave site. Therefore, it276

can not be cleaved by REN, the obtained fluorescence intensity was no significantly277

changed.(see Figure 5, curve 4). The results were in accordance with those obtained by278

electrochemical method. Of course, the performance of the hairpin DNA probe can279

also be monitored with fluorescent method. However, fluorescent method needs280

complex process of fluorescent labeling and purification, which are time consuming,281

labor intensive and cost high. Compared to fluorescent method, the proposed282

electroanalysis has many advantages such as inexpensive instrument, lower detection283

limit and simplicity due to ease of obtaining electrical signal.284


4. Conclusions286

In summary, we have introduced here a novel strategy for development of 287

electrochemical DNA biosensor based on hairpin probe and site-specific DNA288



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cleavage of restriction endonuclease. This biosensor was used to detect DNA species289

related to cymbidium mosaic virus. The results illustrated that the proposed sensor has290

the advantages of higher sensitivity and lower background current. Given the291

simplicity in design of the proposed electrochemical sensor, it is fairly easy to292

generalize this strategy to detect a spectrum of targets. Furthermore, this design could293

be also used to construct novel optical DNA biosensors. So, it might have a promising294

future for investigation of DNA hybridization and would play the potential295

predominance in diagnosis of virus or diseases.296

Acknowledgements297

This work was financially supported by National Basic Research Program of China298

(No.2010CB732403), NSFC (20735002, 40940026, 20775019) of China, the Hi-Tech299

Research and Development Program of China (No. 2006AA09Z168), the National300

Science Foundation of Fujian Province (2009J01023) and the Foundation of Fujian301

Education Department (JA09116).302

303

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Figure captions:354



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Figure 1 Diagram of the procedure for the fabrication of DNA biosensor.355

Figue 2 Impedance spectra (Nyquist plot) of bare GE (a), the S1/MCH modified GE356

(b), the S1/MCH modified GE after being immersed into the BfuCI in reaction buffer 357

solution (c), the S1-T1/MCH modified GE(d) and S1-T1/MCH modified GE after 358

being immersed into the BfuCI in reaction buffer solution (e) in the presence of 2 mM359

[Fe(CN)6]4-/3-

. The biased potential was 0.22V (versus Ag/AgCl) in the frequency360

range of 0.1–105

Hz and the amplitude was 5.0 mV.361

Figure 3 DPV for different target concentrations of T1 sequence (×10-13

mol/L): (f-a)362

background, 1.0, 3.0, 6.0, 10.0, 14.0 and 18.0. Inset: Plot of I versus logarithm of 363

concentration. Error bars =±relative standard deviation.364

Figure 4 DPV of hybridization with different kinds of target DNA. (a) complementary365

sequence; (b) one-base mismatched sequence; (c) non-complementary sequence; All366

the concentrations of target DNA used were 1.8×10−12

M.367

Figure 5 Fluorescent graph of MB hybridizing with different gene fragments. samples368

are MB(curve 1); MB after incubed in REN(curve 2); MB+complementary369

DNA(curve 3); MB+complementary DNA after incubed in REN(curve 4). Buffer 370

solution: NEBuffer 41X (50 mM potassium acetate, 20 mM Tris-aceate, 10 mM371

magnesium acetate, 1 mM dithiothreitol, pH 7.9). λ ex = 521 nm, λ em= 586 nm.372

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Figure 1377

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Figure 2390



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0 4000 8000 12000 16000 20000 24000

4000

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ZRe/Ohm

Z l m / O h m

e

Rs

Ret

Cdl

ac

b d

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Figure 3404

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-0.80 -0.75 -0.70 -0.65 -0.60 -0.55 -0.50 -0.450.10

0.15

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0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

0.15

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E/V

I / A

a

flog(C)/0.1pM

I / A

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Figure 4419

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0.10

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I / A

E/V

a

b

c

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Figure 5434

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540 560 580 600 620 640 6600

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F

1

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nm

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Table 1 Details of the DNA sequences448

Capture probe (S1)a

5′-HS-AAA A GA TCT T CA AAC TGC GGA CAT T AA GAT C-3′

molecular beacon b

5'-TAMRA-GATCTT CAA ACTGCGGACATT AAGATC-DABCYL-3'

Target (T1) 5′- AAT GTC CGC AGT TTG-3′

single-base mismatch (T2) 5′-AAT GTC CAC AGT TTG-3′

non-complementary (T3) 5′- CCG TCA ATT AAG CCA-3′

a The program was used to predict the solution-phase conformation of this sequence.449

This program predicts that under the hybridization conditions adopted for the analysis,450

only one secondary structure (the hairpin one) is thermodynamically stable, confirming451

the suitability of the chosen sequence (Zuker et al., 2003).452

b Tetramethoxyl Rhodamine (TAMRA) as fluorophore, 4-(2-methyl453

on-amino-azobenzene) benzoate (DABCYL) as quencher.454

455

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Documents

A Strategy for Development of Electrochemical DNA Bio Sensors