Structure-Switching Electrochemical Aptasensor for Single-Step and

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Food Safety and Toxicology

Structure-switching Electrochemical Aptasensor for Singlestep and Specific Detection of Trace Mercury in Dairy Product Xinai Zhang, Chenyong Huang, Yanjuan Jiang, Yuxiang Jiang, Jianzhong Shen, and En Han J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03259 • Publication Date (Web): 05 Sep 2018 Downloaded from http://pubs.acs.org on September 12, 2018

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Journal of Agricultural and Food Chemistry

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Structure-switching Electrochemical Aptasensor for Single-step and Specific

2

Detection of Trace Mercury in Dairy Product

3 4

Xinai Zhang,* Chenyong Huang, Yanjuan Jiang, Yuxiang Jiang, Jianzhong Shen, En Han

5

School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, P.R. China

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

*Corresponding author: Dr. Xinai Zhang Tel: +86-511-88780201 E-mail: [email protected]

27 28 29 30 31 32 33

1

ACS Paragon Plus Environment


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Abstract

35

A reagentless and single-step electrochemical aptasensor with separation-free fashion

36

and rapid response is developed for Hg2+ assay in dairy product. Herein, the sensing

37

strategy is established on Hg2+-induced structural transition of the methylene

38

blue-tagged single-stranded DNA (ssDNA) from flexible manner to rigid

39

hairpin-shaped double-stranded DNA (dsDNA), generating improved peak current for

40

Hg2+ assay with detection limit of 0.62 fM. Importantly, the best signal-to-noise ratio

41

value can be obtained by exploiting Au flowers as sensing material and the optimal

42

ssDNA concentration. The proposed sensor also exhibits high selectivity due to the

43

specific thymine-Hg2+-thymine (T-Hg2+-T) coordination chemistry, and can be applied

44

to detect Hg2+ in dairy product. With the use of the electric “signal-on” switch, the

45

electrochemical aptasensor has the advantages of simplicity, ease of operation, high

46

sensitivity and specificity, offering a promising method to assess the safety of dairy

47

product polluted with Hg2+.

48 49 50

Keywords

51

mercury ions, milk, electrochemical aptasensor, simplicity, structure-switching

52

2


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Introduction

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Dairy product is considered as highly valuable food due to its rich nutritional

55

components such as minerals, vitamins and protein. Unfortunately, owing to the

56

increasing content of environmental pollutants with the growth of urban, industrial

57

and agricultural discharges, dairy product is vulnerable to be polluted with heavy

58

metals in its production.1-3 According to the U.S. Environmental Protection Agency

59

(USEPA), mercury is one of the most common heavy metals inducing pollution.4

60

Some investigations show that the presence of mercury even at low concentration

61

could disorder body mechanisms such as kidney failure, brain damage and

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cardiovascular systems destruction.5-8 Therefore, it is essential to develop the

63

analytical techniques for effective monitoring of trace mercury in dairy product.

64

Up to now, some techniques have been reported for mercury assay in dairy

65

product, such as cold-vapor atomic fluorescence/absorption spectrometry,9 inductively

66

coupled plasma mass spectrometry,10,11 high-performance liquid chromatography,12

67

and sensing strategy.13-17 Compared with these analytical techniques, sensing strategy

68

has received extensive interest because of its rapid detection and tremendous

69

versatility. Consequently, several sensors were developed based upon electrochemistry,

70

colorimetry,

71

electrochemical sensor is well recognized as a powerful technique for mercury assay

72

due to its inherent simplicity, high sensitivity and excellent flexibility.

fluorescence,

photoelectrochemistry,

etc.18-23

Among

them,

73

As dairy product is present as complex matrix, it is an urgent task to enhance the

74

specificity and sensitivity of the electrochemical sensor for trace mercury detection. 3



75

At present, aptamers, synthetic oligonucleotides (DNA or RNA) that bind to various

76

target molecules (metal ions, small molecules, proteins, cells, etc.) with high

77

affinity,24,25 have drawn particular interest in sensor design. Owing to their own merits

78

of low cost, specificity and stability,26-30 some aptasensors have been developed for

79

different types of bioassay of Hg2+ (one of most stable and universal inorganic forms

80

lying in mercury contamination).31 Especially, the formation of thymine-Hg2+-thymine

81

(T-Hg2+-T) coordination between Hg2+ and T-rich DNA strands is a widely used

82

approach for Hg2+ determination with good selectivity.32,33 Regarding sensitivity,

83

many attempts have been made in achieving signal amplification and signal output to

84

improve the detectability of the electrochemical sensing strategy. In this respect,

85

redox-labels including methylene blue (MB) and ferrocene (Fc) are considered as

86

promising tags for signal output due to their easy chemical modification and

87

convenient redox potential.34-37 Meanwhile, nanomaterials that are utilized as sensing

88

platform for electrode modification play a crucial role in promoting electron transfer

89

for signal amplification.38-41 Recently, various electrochemical sensors have been

90

developed to significantly facilitate Hg2+ analysis based on nanomaterials and

91

T-Hg2+-T base pairs.42,43 Although sensitive and selective, some of the protocols

92

require multiple washing steps and operations, possibly affecting the reproducibility

93

and stability of the sensors.

94

In response to the shortcomings, we report here an electrochemical aptasensor

95

for single-step detection of Hg2+ in dairy product by utilizing T-Hg2+-T complex

96

without multiple operations and labeling combination. The sensing strategy is simply 4


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established on Hg2+-induced structural transition of MB-labeled single-stranded DNA

98

(ssDNA) from flexible manner to rigid hairpin-shaped double-stranded DNA

99

(dsDNA), which shortens the distance between MB tags and the electrode surface and

100

thus generates improved peak current.35 Moreover, with the use of Au flowers-sensing

101

platform and the optimal ssDNA concentration, the best signal-to-noise ratio can be

102

achieved to improve the sensor performance. When compared with other sensors

103

based on Au flowers,44,45 the proposed aptasensor provides a signal enhancement

104

platform to facilitate high sensitivity. On basis of the electric “signal-on” switch and

105

the specific T-Hg2+-T coordination chemistry, the electrochemical aptasensor can

106

achieve separation-free, simple, selective and sensitive assay of Hg2+, offering a

107

promising tool for Hg2+ analysis in dairy product.

108

Materials and Methods

109

Chemicals and Materials. Mercury nitrate (Hg(NO3)2), 6-mercapto-hexanol

110

(MCH), Tris (2-carboxyethy) phosphine hydrochloride (TCEP) and Nafion were

111

obtained from Sigma-Aldrich. The Hg2+-target ssDNA labeled with 5'-SH and 3'-MB

112

was provided by Sangon Biotechnology Co., Ltd. (Shanghai, China). The sequences

113

were given as follows: 5'-SH-(CH2)6-TTCTTTCTTCGCGTTGTTTGTT-MB-3'.

114

Chloroauric acid (HAuCl4·4H2O), dopamine (DA) and other chemical reagents were

115

obtained from Shanghai Sinopharm Inc. (Shanghai, China). 20 mM Tris-HCl buffer

116

(pH 7.0, containing140 mM NaCl, 1 mM MgCl2 and 5 mM KCl) was utilized as

117

buffer solution. Hexaammine ruthenium(III) chloride (Ru(NH3)6Cl3, RuHeX) was

118

supplied by Sinocompound Catalysts Co. Ltd. Fresh milk was obtained from 5



119

Changjiang Dairy Co., Ltd. (Zhenjiang, China). All chemicals were of

120

analytical-reagent grade. Pure water obtained from Millipore (Milli-Q, 18.2 MΩ·cm)

121

was used to prepare all solutions.

122

Apparatus. A CHI630D workstation (Shanghai CH Instrument Co., China)

123

consisting of a saturated calomel reference electrode (SCE), the modified glassy

124

carbon working electrode (GCE, 3 mm in diameter) and a platinum wire auxiliary

125

electrode was utilized to perform cyclic voltammetry (CV) and square wave

126

voltammetric (SWV) measurements. The morphology of the prepared nanomaterials

127

was characterized by S-4800 scanning electron microscopy (SEM) (Hitachi Co., Ltd.,

128

Tokyo, Japan).

129

Au flowers’ Fabrication. The preparation of Au flowers were as follows.44

130

Firstly, 1% HAuCl4 (100 µL) was added to the mixture solution (1 mL) of 20 mM DA

131

and 20 µL of 0.5% Nafion with stirring for 60 min at room temperature until the color

132

became light red. Subsequently, the resulting solution was purified by centrifuging for

133

10 min at 10,000 rpm to obtain Au flowers. After centrifugal cleaning three times with

134

pure water, the precipitate was dispersed in pure water (1 mL) and kept at 4 °C until

135

use.

136

Preparation of the ssDNA/Au/GCE Aptasensor. The working electrode of

137

GCE was first treated with alumina of different particle sizes (0.3 and 0.05 µm), and

138

then the polished electrode was sonicated in acetone, HNO3 (1:1 v/v), NaOH (50%

139

w/w) and water. Subsequently, Au flowers (10 µL) were dropped onto a pretreated

140

GCE and then dried under infrared light to achieve Au/GCE. When the Au/GCE was 6


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utilized to immobilize T-rich ssDNA, it should be kept clean by rinsing with pure

142

water and drying with nitrogen. Additionally, the sensing platform including

143

PDA-AuNPs/GCE, AuNPs/GCE and PDA/GCE were also prepared to perform the

144

control experiments (PDA: polydopamine, AuNPs: Au nanoparticles). In detail, the

145

bare GCE was immersed in the mixture solution of 20 mM DA and 20 mM HAuCl4,

146

and then 20 cycles of CV measurements were performed between −0.9 and +1.0 V

147

under a scan rate of 20 mV s−1. As a result, the PDA-AuNPs/GCE was fabricated after

148

thoroughly rinsed with water. Using the same method, AuNPs/GCE and PDA/GCE

149

were also prepared for the following assay.

150

Prior to electrode functionalization, Hg2+-target DNA strands were introduced

151

into TCEP (5 mM) in a centrifuge tube, and then reacted for 1 h to reduce the formed

152

disulfide. Subsequently, the mixture was dissolved in Tris-HCl buffer (20 mM, pH 7.0)

153

to obtain 1.0 µM final concentration. Next, 10 µL of ssDNA (1.0 µM) was placed

154

onto the Au/GCE electrode for 4 h at 4 °C to achieve the ssDNA/Au/GCE. After

155

being carefully rinsed with Tris-HCl buffer, the ssDNA-modified Au/GCE was

156

passivated with MCH (10 µL) for 1 h to reduce nonspecific binding effects.

157

Electrochemical Measurement. The ssDNA/Au/GCE was incubated with Hg2+

158

standard solutions or real samples for 1 h at room temperature to form specific

159

T-Hg2+-T complex. After being rinsed with Tris-HCl buffer, the obtained electrode

160

was then subjected to SWV measurements with the potential window from −0.50 to 0

161

V under a step potential of 4 mV, a frequency of 10 Hz, and an apmplitude of 25 mV.

162

Results and Discussion 7



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Mechanism of Electrochemical Sensing Strategy. Figure 1 illustrates the

164

structure of the Hg2+-target ssDNA strands and the application of these DNA strands

165

for single-step electrochemical assay of Hg2+. The T-rich ssDNA strands are

166

composed of the thiol portion (−SH, 5') and MB portion (3'), in which the thiol part

167

anchors the strands on Au flowers-modified GCE (Au/GCE) via Au-S bond, while

168

MB portion as redox label is responsible to generate signal output for Hg2+ detection.

169

The ssDNA strands remain in a flexible manner without Hg2+, and the MB labeled at

170

the distal end is far away from the underlying electrode. Consequently, the redox MB

171

tags can’t effectively exchange electrons with the electrode, producing low

172

electrochemical

173

ssDNA/Au/GCE sensor, the target Hg2+ triggers the structural transition of the flexible

174

aptamer to form a rigid dsDNA via chemical coordination of T-Hg2+-T. As a result, the

175

collisions between the MB tags and the sensor surface increase significantly, leading

176

to enhanced current response of MB (ON state). The current increase is related to

177

Hg2+-induced conformational changes of MB-labeled DNA strands, and thus reflects

178

target Hg2+ concentration in the samples.

signals

(OFF

state).

When

Hg2+

is

incubated

with

the

179

Characteristics of Au Flowers as Sensing Platform. The morphology of Au

180

flowers is characterized using SEM (Figure 2A). As can be seen, Au flowers present

181

flower-like shapes. The forming reason of flower-like structure is due to the fact that

182

nafion acts as the template for AuCl4− reduction and nucleation.

183

To evaluate the advantages of Au flowers over other conventional materials, four

184

different modified electrodes containing Au/GCE, PDA-AuNPs/GCE, AuNPs/GCE 8


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185

and PDA/GCE are applied to the sensor design for 1 nM Hg2+ detection. The

186

evaluation is based on monitoring the signal-to-noise ratio (i/i0, the letter i and i0

187

represent current response with the presence and absence of Hg2+ respectively) of the

188

different sensors.

189

As shown in Figure 2B, Au/GCE exhibits similar SWV peak current to that of

190

PDA-AuNPs/GCE, AuNPs/GCE and PDA/GCE in the absence of Hg2+. This is

191

probably due to the ssDNA structure in a flexible manner that inhibits electron

192

exchange between MB tags and different sensing surfaces. However, Au/GCE

193

exhibits the best SWV peak current with presence of Hg2+, producing the highest i/i0

194

value. The reason might be due to the fact that Au flowers could provide large surface

195

area to load more MB-labeled ssDNA and also have good conductivity to rapid

196

electron transfer rate. The experimental results demonstrate that Au flowers as sensing

197

material can provide obvious superiority over PDA-AuNPs, AuNPs or PDA in

198

electrochemical performance and possess the ability to improve sensitivity.

199

In order to elucidate that, the electro-active surface area of Au flowers-modified

200

GCE is quantitatively detected by recording cyclic voltammetric curves (CVs) (Figure

201

2C). The CV measurements are performed by utilize [Fe(CN)6]3−/4− as redox probes

202

under different potential scan rates. The electro-active surface area of Au/GCE is

203

calculated to be 11.602 mm2 according to the Randles-Sevcik equation:46-48

204

𝑖 = 2.69 × 105 𝐴𝐷1/2 𝑛3/2 𝑣 1/2 𝐶

(1)

205

in which i refers to the redox peak current, A is the electrode area, D represents the

206

diffusion coefficient (at 25 °C, D=6.70×10−6 cm2 s−1), n is the number of electrons

207

transferred in the redox reaction (n=1), v is the scan rate of the CV measurement and 9



208

C is the concentration of the reactant (5 mM Fe(CN)63−/4−). This demonstrates that Au

209

flowers-modified GCE has a larger electro-active surface area in comparison with the

210

bare GCE surface area (7.065 mm2).

211

Optimization of Immobilization Concentration of ssDNA. The effect of the

212

immobilization concentration of ssDNA on the i/i0 value is evaluated to obtain best

213

aptasensor performance. With increasing the ssDNA concentration, the background

214

current increases gradually because of the increasing number of ssDNA loading on the

215

Au/GCE (Figure 3A). With the presence of Hg2+, the concentration of ssDNA at 1.0

216

µM displays the maximum response and produces the highest i/i0 value, which is due

217

to the fact that the combination possibility between the flexible ssDNA and Hg2+

218

decreases at a lower concentration, while a higher concentration partially inhibits the

219

binding because of the steric hindrance resulting from the immobilized ssDNA with

220

higher surface densities. Therefore, 1.0 µM ssDNA is used to prepare the aptasensor.

221

Additionally, the effects of pH condition, incubation time and temperature between

222

ssDNA and Hg2+ on detecting efficiency are also investigated. According to the

223

experimental results (Figure S1), the incubation of the immobilized ssDNA with Hg2+

224

in pH 7.0 Tris-HCl buffer for 60 min at room temperature is selected for the formation

225

of the specific T-Hg2+-T complex.

226

Meanwhile, the density of ssDNA on Au/GCE surface is measured by the

227

chronocoulometric method and the result is shown in Figure 3B. According to the

228

Cottrell’s equations49,50 listed below, the surface density of the ssDNA on Au/GCE

229

surface is calculated to be 2.77×1012 molecules per cm2. 10


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𝑄𝑠𝑠𝐷𝑁𝐴 −𝑄𝑀𝐶𝐻

230

𝛤𝑅𝑢 =

231

𝛤𝑠𝑠𝐷𝑁𝐴 = 𝛤𝑅𝑢 (𝑧/𝑚)𝑁𝐴

(2)

𝑛𝐹𝐴

(3)

232

where ΓRu is the amount of the redox marker restricted near the sensing surface

233

(mol/cm2), QssDNA and QMCH are the surface charges (C), n represents the electron

234

number in the reaction, F refers to the Faraday constant, A is the Au/GCE area (cm2),

235

ΓssDNA is the surface density of DNA, z represents the charge of the redox marker, m is

236

the base numbers in DNA, and NA represents Avogadro’s number.

237

Hg2+ Analysis. Under the optimized experimental parameters, the proposed

238

aptasensor is utilized to detect Hg2+ with different concentrations in Tris-HCl buffer.

239

As seen in Figure 4A, the peak current increases gradually with the increasing Hg2+

240

concentration. The Δi linearly depends on the logarithm of Hg2+ concentration ranging

241

from 1 fM to 1 nM (R2=0.994) (Inset). The limit of detection (LOD) corresponding to

242

S/N of 3σ is 0.62 fM. The LOD of the proposed sensor is better or comparable to the

243

other methods reported previously.25,31-33,42,43 Additionally, when compared with the

244

DNA sensors based on Au nanomaterials,51,52 the proposed aptasensor can achieve the

245

improvement of electrochemical performance, which is ascribed to Au flowers as

246

sensing platform and the “signal-on” format with increased signal gain.

247

Selectivity, Reproducibility and Stability. In order to evaluate the selectivity of

248

the aptasensor, Pb2+, Ca2+, Cu2+, Cd2+, K+ and Na+ are selected as the possible existing

249

metal ions to be analyzed. As seen in Figure 4B, the incubation of the control metal

250

ions (10 nM, 10 times Hg2+ concentration) respectively with the aptasensor has no

251

obvious difference in current response in comparison to that of the blank assay

11



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(without Hg2+). However, the existence of only Hg2+ leads to an obvious increase in

253

current response relative to other interfering substrates. Besides, the aptasensor is

254

employed for Hg2+ detection in the coexistence of other metals. When other metal ion

255

is introduced into Hg2+ solution, there is almost no effect on the current response for

256

Hg2+ assay (Figure 4C). The results demonstrate that the specificity of the present

257

work is acceptable, which is ascribed to the excellent selectivity of thymine base

258

toward Hg2+ to form the strong T-Hg2+-T complex.

259

Furthermore, the reproducibility of the aptasensor is evaluated by using six

260

prepared electrodes to analyze 0.01 nM Hg2+. Under the same conditions, the relative

261

standard deviation (RSD) of the determination is 7.6% (n=6) with the six electrodes,

262

indicating good reproducibility of the sensor. In addition, the long-term stability of the

263

proposed

264

ssDNA/Au/GCE-based aptasensor at 4 °C and assessing every 2~3 days. After a

265

longer storage for 10 days, the SWV peak current could remain 92.6% of the initial

266

value, indicative of good stability.

aptasensor

is

evaluated

on

a

20-d

period

by

storing

the

267

Real-sample Analysis. The developed method is utilized for Hg2+ assay in pure

268

fresh milk to investigate the practical application of the aptasensor. The dairy product

269

samples is prepared as follows: 0.10 mL milk is dissolved into 10 mL pH 7.0 Tris-HCl

270

buffer, and then 10 fM, 100 fM, 1 pM, 10 pM and 100 pM concentration of Hg2+ is

271

respectively spiked into milk under stirring. Subsequently, the as-prepared samples

272

are analyzed by utilizing the standard addition method and the results are exhibited in

273

Table 1. As can be seen, the obtained recoveries range from 88.6% to 115.2%, 12


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indicative of the favorable reliability of the electrochemical strategy.

275

Moreover, in comparison with SWV current response for the analysis of 10 fM

276

Hg2+ only (Figure 5A,C), there is no obvious difference of peak current for 10 fM

277

Hg2+ assay in the sample matrices (containing mineral, protein, fat, vitamins,

278

microorganisms, etc.) of pure fresh milk (Figure 5B,D). The results demonstrate that

279

the exhibiting interferences have no remarkable effect on Hg2+ determination,

280

indicative of the suitability of the aptasensor for Hg2+ assay in dairy product.

281

In conclusion, in the current study, the electrochemical sensor is proposed for

282

Hg2+ assay on basis of the formation of specific T-Hg2+-T coordination chemistry and

283

the structural transition of the MB-labeled ssDNA with T-rich sequence. Compared

284

with the conventional aptasensors for Hg2+ assay, the developed strategy could

285

achieve greatly enhanced sensitivity due to the use of Au flowers as sensing platform

286

and the electric “signal-on” switch with increased signal gain. Overall, the

287

electrochemical aptasensor gives a useful protocol with simplicity, excellent

288

sensitivity and selectivity, and provides a valuable tool for evaluating trace mercury in

289

dairy product.

290

Acknowledgements

291

We would like to thank the technicians in Yangzhou University for their help in

292

nanomaterial characterization.

293

Funding source

294

This work was supported by the National Natural Science Foundation of China

295

(21205051), and Project Funded by the Priority Academic Program Development of 13



296

Jiangsu Higher Education Institutions (1033000006).

297

Supporting Information. Optimization of pH condition, incubation time and

298

temperature between ssDNA and Hg2+ supplied as Supporting Information.

299

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22


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476


Figure captions

477 478

Figure 1. Illustration of the single-step and specific detection of Hg2+ by using Au

479

flowers-based electrochemical aptasensor. Lower left: conformational change of

480

single-stranded DNA (ssDNA) upon binding to Hg2+.

481 482

Figure 2. (A) Scanning electron microscopy (SEM) image of Au flowers. Inset: The

483

photograph for color change of the mixture of 1% HAuCl4, 20 mM dopamine (DA)

484

and 0.5% Nafion before (a) and after (b) stirring for 1 h, and the resulting Au flowers

485

dispersed in deionized water (c). (B) Comparison of the signal to noise ratio (i/i0, i

486

and i0, respectively, correspond to the square wave voltammetric (SWV) peak current

487

with the presence and absence of Hg2+) of the sensor for 1 nM Hg2+ detection based

488

on different sensing platform: Au/GCE, PDA-AuNPs/GCE, AuNPs/GCE and

489

PDA/GCE. (C) CV of Au flowers-modified GCE in 5 mM [Fe(CN)6]3−/4− at different

490

scan rates from 10 to 250 mV s−1. Insets show the linear relations of the Au

491

flowers-modified GCE with the anodic and cathodic peak current against the square

492

root of scan rate.

493 494

Figure 3. (A) Effects of the immobilization concentration of ssDNA on signal to

495

noise ratio (i/i0, i and i0, respectively, correspond to the SWV peak current with the

496

presence and absence of Hg2+) of the sensor for 1 nM Hg2+ detection based on

497

Au/GCE as sensing platform. (B) Chronocoulometric curves for Au/GCE modified

498

with (a) 6-mercapto-hexanol (MCH)/ssDNA and (b) MCH in 20 mM Tris-HCl buffer 23



499

(pH 7.0) with the presence of 50 µM hexaammine ruthenium(III) chloride (RuHex).

500

Redox charges of RuHex confined near the electrode surface are obtained from

501

chronocoulometric intercepts at t=0.

502 503

Figure 4. (A) SWV responses for the sensor toward different concentrations of Hg2+

504

(a) blank, (b) 1 fM, (c) 10 fM, (d) 100 fM, (e) 1 pM, (f) 10 pM, (g) 100 pM, (h) 1 nM

505

(Inset: corresponding calibration curves). The specificity of the electrochemical

506

sensor toward 1 nM Hg2+ against 10 nM Pb2+, 10 nM Ca2+, 10 nM Cu2+, 10 nM Cd2+

507

10 nM K+ and 10 nM Na+: (B) for individual metal ion alone and (C) Hg2+ +

508

co-existed metal ion.

509 510

Figure 5. The electrochemical aptasensor was used for the assay of (A,C) 10 fM Hg2+

511

only and (B,D) 10 fM Hg2+ in sample matrices of pure fresh milk, respectively.

512

24


Page 24 of 36

Page 25 of 36

513

514


Table 1. Determination Results and Recoveries of Pure Fresh Milk Samples

a

sample

detected

spiked

founda

recovery

no.

(pM)

(pM)

(pM)±SD

(%)

1

no detected

0.01

0.0108±0.0006

108

2

no detected

0.1

0.0886±0.0030

88.6

3

no detected

1.0

0.9570±0.0560

95.7

4

no detected

10

9.020±0.5520

90.2

5

no detected

100

115.2±5.484

115.2

Number of samples analyzed was 5.

515

25



516

Figure 1

517 518

26


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Page 27 of 36

519


Figure 2

520 521

27



522

Figure 3

523 524

28


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525


Figure 4

526 527

29



528

Figure 5

529 530

30


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531


Table of Contents Graphic

532 533

31



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Structure-Switching Electrochemical Aptasensor for Single-Step and

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