Multiple Signal Amplified Electrochemiluminescent Immunoassay for

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Multiple Signal Amplified Electrochemiluminescent Immunoassay for Hg2+ Using Graphene-coupled Quantum Dots and Gold Nanoparticles Labeled Horseradish Peroxidase Fudong Cai, Qing Zhu, Kang Zhao, Anping Deng, and Jianguo Li Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b00690 • Publication Date (Web): 23 Mar 2015 Downloaded from http://pubs.acs.org on April 1, 2015

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Environmental Science & Technology

Multiple

Signal

Amplified

Electrochemiluminescent

Immunoassay for Hg2+ Using Graphene-coupled Quantum Dots and Gold Nanoparticles Labeled Horseradish Peroxidase

Fudong Caia, b, Qing Zhua, b, Kang Zhaoa, b, Anping Denga, b*, Jianguo Lia, b*, *

a

*

College of Chemistry, Chemical Engineering & Materials Science, Soochow University, Suzhou 215123, China

b

The Key Lab of Health Chemistry & Molecular Diagnosis of Suzhou, Suzhou 215123, China

* Correspondence authors: J.G. Li, Telephone: +86 51265882195, Fax: +86 51265882195, E-mail address: [email protected]; A.P. Deng, Telephone: +86 51265882362, Fax: +86 51265882362, E-mail address: [email protected]

ACS Paragon Plus Environment 1


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1

ABSTRACT: A multiple signal amplification strategy was designed for ultrasensitive

2

competitive immunoassay for Hg2+. This strategy was achieved using graphene

3

conjugated with large amount of CdSe quantum dots for enhancing the basal signal,

4

gold nanoparticles (AuNPs) labeled enormous horseradish peroxidase (HRP), which

5

consumed the coreactant H2O2 generated in situ. The immunosensor was constructed

6

by

7

chloride)-graphene-CdSe composites (PDDA-GN-CdSe), and a strong ECL signal

8

would be obtained. When immunosensor immersed in antibody-AuNPs-HRP

9

composites, the ECL signal would greatly decrease, which was ascribed to the bound

10

enzyme on the electrode surface. The self-produced coreactant H2O2 is consumed by

11

OPD in the presence of enzyme, effectively decreasing the ECL intensity from the

12

quantum dots. The Hg2+ in solution and the corresponding coating antigen would

13

compete for the limited antibody, thus the ECL intensity is linear to the logarithm of

14

the mercury (II) concentration from 0.2 to 1,000 ng mL−1 and detection limit of 0.06

15

ng mL−1. The immunoassay exhibited good stability, accuracy and acceptable

16

reproducibility, indicating a promising approach for the detection of trace mercury

17

and other small molecular compounds in environmental samples.

18

Keywords: Electrochemiluminescence; Competitive immunoassay; Mercury(II) ion;

19

Multiple Signal Amplification; CdSe quantum dots; Graphene; Gold nanoparticles

immobilizing

coating

antigen

on

the

poly

(diallyldimethylammonium

20


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21

1. INTRODUCTION

22

Nowadays, mercury is widely considered to be one of the most dangerous

23

pollutants and highly toxic element in pollution of the environment.1 Mercury in the

24

environment exists in a variety of forms, such as mercuric ion (Hg2+), mercurous ion

25

(Hg+), mercury sulfide (HgS),2 methylmercury (CH3Hg+),3 ethylmercury (C2H5Hg+),

26

and phenylmercury (C6H5Hg+). The study of mercuric ion is particularly important

27

because mercuric ion (Hg2+) is one of the most stable forms in mercury pollution.4, 5

28

The mercury in body may result in brain damage and kidney failure,6 which seriously

29

threaten people's health. Currently, various methods for detecting Hg2+ have been

30

designed, such as cold vapor atomic absorption spectrometry (CV-AAS),7 atomic

31

emission spectroscopy,8 fluorescence spectrometry,9, 10 enzyme-linked immunosorbent

32

assays (ELISA)11 and high performance liquid chromatography (HPLC).12 Besides,

33

specific conjugation of the Hg2+ with two thymine bases (T) opened up a new path for

34

detection of Hg2+.13 Although these techniques have some advantages in the

35

determination of Hg2+, most of them need expensive and cumbersome instruments

36

and require complicated preparation of sample, which hinder its easy applicability to

37

real samples.14 Therefore, it is urgent to establish a simple, rapid and inexpensive

38

approach for monitoring mercury residues and pollutions.

39

Compared with above methods, electrochemiluminescence (ECL) would be a

40

promising method on account of its advantages of simple sample preparation process,

41

high sensitivity and low cost. Usually, the ECL signals can be obtained from the

42

luminophores such as Ru(bpy)32+, luminol, semiconductor nanocrystals and so on.15-18

43

Quantum dots (QDs), a common nanocrystals, possess the properties of

44

nano-materials, such as surface effect, small size effect, quantum effect, narrow

45

emission spectra and optical properties.19, 20



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Gold nanoparticles (AuNPs) have excellent electrical conductivity, which would

47

greatly accelerate the electron transfer and improve the electrochemical reaction

48

efficiency.21 Besides, owing to the large effective surface area and the special

49

size-depended physical and chemical properties, AuNPs could act as the substrate to

50

load a large number of labels.21 With the help of electrostatic interaction, protein

51

could conjugate well with AuNPs and be modified on the surface of electrode.

52

Currently immunoassays have also been applied for detecting heavy metals.11, 22, 23 To

53

pursue highly selectivity, monoclonal antibody that binds specifically to mercuric ions

54

was applied as a creative mode. 6-mercaptonicotinic acid (MNA), a new ligand with

55

double functional group, links mercury and carrier protein to produce the

56

mercury-MNA-protein conjugates. The conjugates could further generate coating

57

antigen and immune antigen. According to our previous literature,24 the mAb

58

exhibited a high affinity recognition and high specificity of Hg2+.

59

Graphene (GN), a thin film of carbon atoms in a honeycomb crystal lattice,

60

possesses magical optical, electrical conductivity, mechanical, thermal and chemical

61

properties.25, 26 These features and some other features make graphene apply in many

62

fields, such as the field of electrochemiluminescence. However, GN is hydrophobic

63

and tends to agglomerate irreversibly through Van der Waals interaction.27 To avoid

64

this agglomeration of graphene sheets, great efforts have been devoted to

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functionalize GN via chemical modification with dispersive reagents, such as

66

surfactant and polymers.28 Poly(diallyldimethylammonium chloride) (PDDA), a

67

watersoluble quaternary ammonium and cationic polyelectrolyte,29 has been used to

68

noncovalently functionalize graphene oxide. The use of PDDA, the stabilizer and

69

reducing agent, resulted in well dispersion of graphene in aqueous solution and the

70

successful reduction of GO.30 Interestingly, the reduced graphene functionalized by


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the positively charged PDDA could anchor negatively charged materials for

72

manufacturing multifunctional composites.30 Meanwhile, the thioglycolic acid (TGA)

73

modified CdSe QDs we used are semiconductor nanoparticles containing negatively

74

charged thioglycolic.31, 32

75

In this work, we have designed a sensitive and rapid ECL immunoassay for

76

detecting Hg2+ based on PDDA-GN-CdSe composite and AuNPs marker. This novel

77

sensing technology possessed some significant advantages. Firstly, the PDDA-GN

78

have good stability, bioactivity and high specific surface areas, which could assemble

79

more CdSe QDs and provide an effective matrix for antigen immobilization. Secondly,

80

the AuNPs were creatively used to couple with horseradish peroxidase (HRP), which

81

significantly consumed the H2O2 generated in situ and amplified the ECL quenching.

82

Besides, in the competitive immunoassay, Hg2+ in detection solution would compete

83

with the modified coating antigen for the limited AuNPs-labeled monoclonal antibody

84

(mAb), which brought about the high specificity. This sensing system achieved a low

85

detection limit of 0.06 ng mL 1, which was lower than the WHO and US EPA defined

86

toxicity level of Hg2+ in drinking water (6 ppb and 2 ppb).33,

87

advantages indicated that this sensor would be an alternative approach for the analysis

88

of Hg2+ in environmental water samples.

89

2. EXPERIMENTAL SECTION

90

2.1. Chemicals and materials.

−

34

These above

91

Cadmium chloride (CdCl2·2.5H2O, 99%), Se powder (99.95%), thioglycollic

92

acid (TGA), sodium borohydride (NaBH4, 96%), isopropyl alcohol (99.7%),

93

Tris(hydroxymethyl) aminomethane, potassium nitrate (KNO3), graphite powder,

94

glutaraldehyde solution (GLD, 25%), chloroauric acid (HAuCl4·4H2O, 47.8%),



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95

trisodium citrate, o-phenylenediamine (OPD), poly (diallyldimethylammonium

96

chloride) (PDDA) and horseradish peroxidase (HRP), all above reagents were

97

purchased from Sinopharm Chemical Reagent Co., Ltd (China, www. Sinoreagent.

98

com). Aluminum oxide polishing powder (Al2O3, 1.0, 0.3 and 0.05 µm) was obtained

99

from Tianjin Aidahengsheng Technology Co., Ltd (Tianjin, China). Bovine serum

100

albumin (BSA) and ovalbumin (OVA) were purchased from Sigma-Aldrich Co., Ltd

101

(USA, www. sigmaaldrich. com/united-states. html). Mercuric chloride (HgCl2) and

102

6-mercaptonicotinic acid (MNA) were obtained from Sigma Chemical Co. (St. Louis,

103

MO, USA).

104

0.1 M pH 7.4 phosphate buffer saline (PBS) containing of NaCl (100 mM),

105

Na2HPO4 (6.4 mM) and KH2PO4 (1.0 mM) was used as a washing buffer. ECL

106

detection buffer was prepared by 0.1 mol L-1 Tris-HCl buffer containing 0.1 M KNO3

107

and 1.5 mM OPD. Mercury stock solution (1 mg mL-1) was prepared by dissolving

108

6.77 mg HgCl2 in 5 mL of 2% (v/v) HNO3 and kept at 4 °C. All the chemicals and

109

reagents were of analytical reagent grade. All aqueous solutions were prepared with

110

sub-boiling doubly distilled water.

111

2.2. Apparatus.

112

The ECL emissions were recorded by using a MPI-A multifunctional

113

electrochemical analytical system (Xi’An Remax Electronic Science & Technology

114

Co. Ltd, Xi An, China) in the ECL detection buffer, and the working potential was 0 ~

115

-1.3 V with the voltage of the photomuitplier tube (PMT) set at -650 V. The

116

experiment applied a conventional three-electrode system which was composed of a

117

modified glass carbon working electrode (GCE, φ= 3 mm), an Ag/AgCl reference

118

electrode (KCl saturated) and a Pt wire counter electrode.

119

UV-vis absorption spectrum was carried out on Agilent 8453 UV-vis


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photospectrometer (Agilent Co., America). Electrochemical impedance spectroscopy

121

(EIS) was recorded on a RST electrochemical working station (Suzhou Risetest

122

Instrument Co., Ltd., China, www. rst9999.com). The High Resolution Transmission

123

electron microscopy (HRTEM) images were captured by a Tecnai G2 F20 S-TWIN

124

200KV (FEI Co., U.S.A).

125

2.3. Synthesis of the CH3Hg-MNA coating antigen and monoclonal antibody.

126

Firstly, we have prepared mAb and CH3Hg-MNA coating antigen. The

127

preparation process have been introduced in our previous literature.24 As reported in

128

the literature, the mAb exhibited high affinity recognition of mercury ion although

129

using CH3Hg-MNA-BSA as immunogen.24

130

2.4. Preparation of AuNPs and Ab-AuNPs-HRP composite.

131

The AuNPs were prepared by a well-established citrate-reduction method.35 The

132

prepared AuNPs provided a matrix to carry HRP and antibody, which could keep the

133

activity of proteins and enhance the quenching of ECL emission. For prepartion of

134

HRP and Ab combined with AuNPs, 100 µL HRP and 10 µL 5 µg mL-1 Ab were

135

simultaneously added to AuNPs solution. The Ab-AuNPs-HRP composite was

136

obtained via the -Au-S- bonds or -Au-NH- bonds by simply shaking at 4 °C

137

overnight.36 Afterwards, the mixture was centrifuged for 10 min at 10000 rpm, and the

138

supernatant was discarded. The collected compound was redispersed in doubly

139

distilled water and stored at 4 °C.

140

2.5. Synthesis of PDDA-GN and PDDA-GN-CdSe composite.

141

According to the modified Hummers method, 30 we prepared graphene oxide

142

(GO) by graphite powder. Afterwards, 0.2 mL 20% PDDA was added into 20.0 mL

143

0.5 mg mL-1 the homogeneous GO suspension. After sonicated for 30 min, the

144

mixture was heated for 8 min by household microwave oven.30 The above solution



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145

was centrifuged and washed twice with doubly distilled water to remove the

146

redundant PDDA. The final precipitate was dried in vacuum at 68 °C for 10 min and

147

was redispersed in doubly distilled water to obtain 1 mg mL-1 PDDA-GN suspension.

148

Thioglycolic acid (TGA) modified CdSe QDs were synthesized on the basis of

149

previously reported method.21 The PDDA-GN-CdSe complex was prepared as follows:

150

firstly, 1 mL of the prepared QD solution was concentrated by mixing with an equal

151

volume of isopropyl alcohol and centrifuged at 10,000 rpm for 5 min. After separating

152

the precipitation, the precipitation was dissolved in 25 µL water. Afterwards, the

153

PDDA-GN mixed with the concentrated QDs in the same volume, and sonicated for

154

30 min, the resulting compound of PDDA-GN-CdSe stored at 4 °C for use.

155

2.6. Construction of the immunosensor.

156

A glassy carbon electrode (GCE) was successively polished with 0.3 and 0.05

157

μm alumina powder, followed by sonicating respectively in ethanol and distilled

158

water for 5 min and dried with N2. Then the surface of the working electrode was

159

covered by 10 µL PDDA-GN-CdSe and dried at room temperature (RT).

160

Subsequently, 10 µL of 0.025% chitosan was dripped on the surface and dried at RT.

161

Then, 10 µL of 2% glutaraldehyde activated the chitosan film for 1 h and the

162

electrode was incubated by 10 µL of 15 µg mL-1 coating antigen for 1 h at RT and

163

overnight in a refrigerator. 10 µL 5% BSA were applied to block the remaining active

164

groups and eliminate nonspecific adsorptions, and then rinsed with PBST.

165

2.7. Detection with electrochemiluminescence.

166

5 µL of different concentrations of standard mercury solutions were mixed with 5

167

µL of Ab-AuNPs-HRP bioconjugates to obtain the incubation solution. Then the

168

incubation solution was coated on the ECL immunosensor at 37 °C for 1 h, followed

169

by washing with PBST (Scheme 1). Eventually, the fabricated GCE was scanned in


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170

ECL detection buffer and the ECL signals related to the Hg2+ concentrations were

171

measured.

172

173

Scheme 1. Illustrative ECL detection mechanism for Hg2+ based on GCE/PDDA-GN-

174

CdSe/chitosan/Ag/Ab-AuNPs-HRP.

175

3.RESULTS AND DISCUSSION

176

3.1. Characterization of PDDA-GN and PDDA-GN-CdSe.

177

The characterization of the products were shown in Figure 1. Figure 1A shows

178

the HRTEM image of PDDA-GN sheets with transparent flakelike shape. The

179

corrugated parts and folded parts were induced by the electron repelling between

180

layers. The HRTEM of PDDA-GN-CdSe composites were shown in Figure 1B, from

181

which the dark particles on the surface of PDDA-GN can be clearly observed. Free

182

QDs was not found, which indicated complete deposition of the CdSe QDs. The

183

actual size of the deposited CdSe QDs was about 4 nm, which was shown in Figure

184

1C. The crystalline features of CdSe QDs could be clearly observed from the inset in

185

Figure 1C.



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186

187

Figure 1. (A) HRTEM image of PDDA-GN, (B) HRTEM image of PDDA -GN-CdSe

188

composite, (C) HRTEM image of CdSe QDs and (D) UV–vis absorption spectra: (a)

189

CdSe QDs, (b) PDDA-GN-CdSe composite and (c) PDDA-GN.

190

For comparison, the UV-vis spectrum of CdSe QDs (curve a in Figure. 1D),

191

PDDA-GN (curve c in Figure. 1D) and PDDA-GN-CdSe composites (curve b in

192

Figure. 1D) were also plotted. Previous work has reported CdSe QDs absorption peak

193

could not be obtained after QDs were anchored to CNTs, which was ascribed to the

194

wide absorption band of the CNT that could largely shield the absorption peak of the

195

QDs. Only if QDs densely monodispersed onto the CNTs , could the QDs absorption

196

band be seen.37 However, we could clearly find the adsorption peak of CdSe in this

197

study (curve b, Figure 1 D), which suggested a dense dispersion of CdSe QDs onto


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198

the PDDA-GN sheets. Moreover, no obvious change of the absorption peak of the

199

QDs was observed, which demonstrated that no QDs agglomerated on the PDDA-GN

200

sheets and the conclusion was consistent with HRTEM observations.

201

3.2. Characterization of AuNPs.

202

Figure 2A was the UV–vis absorption spectra of the AuNPs, and the absorption

203

band occurred at 521 nm. The prepared AuNPs dispersed uniformly without

204

aggregation, which was confirmed from Figure 2B. The AuNPs have an average

205

diameter of 16 nm, which would supply more active binding sites for the combination

206

of antibody and HRP.

207 208

209

Figure 2. UV–vis absorption spectrum (A) and HRTEM (B) of AuNPs. 3.3. EIS Characterization of Immunosensor.

210

The electrochemical impedance spectroscopy (EIS) of the immunosensor could

211

provide more information about the stepwise modification processes in 0.1 mol L 1

212

KCl containing 5 mmol L 1 [Fe (CN)6]3-/4-. The EIS is composed of two portions: the

213

linear portion and the semicircle portion. The semicircle portion reflected the

214

electron-transfer resistance (Ret) at high frequencies, and a linear portion represented

215

the diffusion process at low frequencies. Figure 3 revealed that the bare GCE

−

−



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216

exhibited a really small Ret (curve a). After the bare electrode was modified with

217

CdSe QDs, the Ret value increases (curve c) due to the increasing impedance.

218

However, when coating the PDDA-GN-CdSe composites film, the Ret value

219

decreased

220

electron-transfer between buffer solution and the electrode surface. The modification

221

of chitosan after PDDA-GN-CdSe composite led to a much larger Ret (curve d),

222

because it was not conductive and blocked the electron transfer. Similarly, coating

223

antigen and BSA could also form the additional obstacles to keep the electron from

224

transferring to the electrode surface (curve e and f). These EIS results demonstrated

225

that these substances successfully modified on the surface of electrode.

dramatically

(curve

b),

indicating

PDDA-GN

accelerated

the

226

227 228

Figure 3. EIS of (a) bare glassy carbon electrode, (b) GCE/PDDA-GN-CdSe, (c)

229

GCE/CdSe, (d) GCE/PDDA-GN-CdSe/chitosan, (e) GCE/PDDA-GN-CdSe/chitosan

230

/Ag and (f) GCE/PDDA-GN-CdSe/chitosan/Ag/BSA in 0.1 mol L−1 KCl solution

231

containing 5 mmol L−1 [Fe (CN)6]3−/4−.

232

3.4. Electrochemical and ECL behavior of Immunosensor.


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233 234

Figure 4. ECL curve and cyclic voltammogram (inset) of the detection Hg2+ at 10 ng

235

mL−1 in oxygen-saturated pH 9.0 Tris–HCl buffer containing 0.1 mM KNO3 and 1.5

236

mM OPD at 100 mV s−1.

237

The curve in Figure 4 (inset) showed the CV of the detection electrode, two

238

reduction peaks appeared at -0.72 V and -0.92 V, due to the reduction of saturated

239

oxygen in the buffer solution and TGA-CdSe QDs, respectively.38 Dissolved oxygen

240

around electrode surface was reduced into H2O2, which has been extensively used as a

241

co-reactant of cathodic ECL of QDs. The self-produced H2O2 reacted with the

242

electron-injected QD ● to generate excited QDs at −0.92 V . Then an intensive ECL

243

emission peak could be observed at − 1.2 V (Figure 4). According to the above

244

explanation and our previous study,39 the ECL processes could be expressed below:

*

−

245

O2 + 2e− + 2H2O → H2O2 + 2OH−

(1)

246

CdSe + e− → CdSe−●

(2)

247

H2O2 + 2CdSe−● → 2CdSe* + 2OH−

(3)

248

CdSe* → CdSe + hν

(4)

249

OPD + H2O2

HRP →  DAP + H2O

(5)



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250

The addition of either HRP or OPD in the system causes negligible changes in

251

ECL intensity of the excited CdSe QDs. However, in presence of the HRP, the ECL

252

intensity of the immunosensor decreased greatly upon addition of OPD in the

253

detection buffer which resulted in the consumption of H2O2 (eq.5), a critical

254

coreactant produced from the dissolved O2 on the electrode surface,38, 39 thus resulting

255

in a quenching effect. Previous studies have shown that HRP and hydroquinone (HQ)

256

can quench excited-states of QDs,38, 39 and the OPD is equivalent to HQ in consuming

257

the H2O2.40

258

3.5. Optimization of immunoreaction conditions.

259

To build optimal conditions for ECL detecting Hg2+, the amounts of coating

260

antigen, the concentrations of antibody, and the volumes of HRP were investigated

261

systematically. The amounts of coating antigen highly influence the quenching

262

efficiency of ECL. The ECL quenching increased drastically when the amount of

263

coating antigen increased. However, when it was beyond 20 µg mL-1 (Figure 5A), the

264

ECL quenching increased slowly and tended a plateau, implying that the coating

265

antigen immobilized on the electrode surface was enough for immunoassay. Thus, 20

266

µg mL-1 of coating antigen was chosen to assemble the immunosensor. In the

267

competitive immunoassays, the concentrations of antibody not only affect the

268

quenching efficiency of ECL but also affect the quantification of Hg2+. The quenching

269

efficiency of ECL would be low if the concentration of antibody is low. However, it

270

could not be too high, because the Hg2+ would conjugate with the excess antibody.

271

Figure 5B illustrated the ECL quenching versus different concentrations of antibody

272

(1 µg mL-1, 2 µg mL-1, 4 µg mL-1, 5 µg mL-1 and 8 µg mL-1). When the concentration

273

of antiody was beyond 5 µg mL 1, the ECL quenching slowly decreased, due to large

274

amouts of antibodies occupied the limited active binding sites, which resulted in

−


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275

fewer HRP labeled on AuNPs. Therefore, 5 µg mL-1 was selected as the optimal

276

concentration of the mAb to combine with AuNPs.

277

278

Figure 5. Optimizations of concentrations of (A) coating antigen and (B) antibody,

279

and (C) volumes of 1mg mL−1 HRP for immunoreactions and enzymic catalytic

280

reaction.

281

Due to the HRP could catalyze OPD to consume the coreactant H2O2, the amount

282

of HRP is one of the most important factors affecting the quenching efficiency of ECL.

283

When the volume of HRP beyond 150 µL (C[HRP] = 1.0 mg mL-1), the ECL quenching

284

slowly increased and tended a plateau, revealing the optimal volume of HRP. Thus

285

150 µL HRP was chosen for preparation of the Ab-AuNPs-HRP composite.

286

3.6. ECL detection of Hg2+ with the proposed immunoassay.

287

Under the optimized experimental conditions, competitive immunoassay was

288

applied for detecting Hg2+. Figure 6 showed the relation between the ECL intensity

289

and Hg2+ standard concentrations. When Hg2+ standard solution was mixed in the

290

incubation solution, the ECL intensity increased linearly with the logarithm of Hg2+

291

concentrations in range from 0.2 to 1,000 ng mL 1 (inset in Figure 6). The regression

292

equation was y = 2564logCHg2+ (ng mL-1) + 3851.5 with a correlation coefficient R of

293

0.9915. Compared with the previous literatures for detecting Hg2+, this study has a

294

wider linear range and a lower LOD (LOD= 0.06 ng mL 1), indicating that the

−

−



295

Page 16 of 35

proposed immunosensor has an excellent analytical performance.

296

297

Figure 6. Cyclic ECL curves of GCE/PDDA-GN-CdSe/chitosan/Ag/Ab-AuNPs-HRP

298

for Hg2+ detection at (a) 1000 ng mL−1, (b) 100 ng mL−1, (c) 10 ng mL−1, (d) 5 ng

299

mL−1, (e) 1 ng mL−1, (f) 0.5 ng mL−1 in oxygen-saturated pH 9.0 tris-HCl buffer

300

containing 0.1 M KNO3 and 1.5 mM OPD. Inset: linear calibration curve for Hg2+

301

detection (n = 3).

302

3.7. Specificity, repeatability and stability of the Immunosensor.

303 304

Figure 7. (A) Selectivity of the developed immunosensor for Hg2+ detection over

305

other metal ions. The concentrations of the metal ions are 100 ng mL-1. (B)


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306

Continuous cyclic scans of immunosensor formed at 0.5 ng mL−1, 1 ng mL−1, 10 ng

307

mL−1, 100 ng mL−1 and 500 ng mL−1 Hg2+ standard solutions, respectively. Other

308

conditions are the same as in Figure 4.

309

To evaluate the selectivity and specificity of the present immunosensor, several

310

other metal ions including Ba2+, Mg2+, Cu2+, Pb2+, Ni2+, Ca2+, Na+, K+, Cd2+ at a

311

concentration of 100 ng mL-1 were tested under the same conditions as in the case of

312

Hg2+. As shown in Figure 7A, the effects of other metal ions were neglectable. These

313

results ascribed to the completely exposition of mercury on the MNA moiety of the

314

molecular structure of CH3Hg-MNA, making it very likely that the metal itself was

315

recognized as a distinct entity by the immune system.24 In summary, the

316

immunosensor had the favorable selectivity for the determination of Hg2+.

317

Repeatability and stability of the immunoassay for Hg2+ was investigated with

318

inter and intra-assay precision. The inter-assay precision of five immunosensors

319

fabricated independently was evaluated by continuous cyclic scans for detecting 50 ng

320

mL-1 Hg2+. All electrodes exhibited homologous ECL responses and the relative

321

standard deviation (RSD) was 3.0%, indicating a more extensive future of application.

322

The stability of the immunosensor was also evaluated by executing 5 reduplicative

323

measurements for the determination of different concentrations of Hg2+. As shown in

324

Figure 7B, the RSD was in the range of 0.9%~ 2.1%, suggested that the proposed

325

immunoassay had excellent stability.

326

3.8. Application of real samples analysis.

327

Several collected environment water samples mixed with Hg2+ were tested using

328

the proposed immunosensor to test its practical application. First, the water samples

329

collected from Dushu Lake and a local river (Suzhou, China), were filtered by 0.45

330

µm membrane to get rid of some insolubles. All collected water samples didn't found



Page 18 of 35

331

the mercury residue by the immunsensor, thus they could be used for blank samples.

332

The recoveries of samples were carried out by spiking different concentrations of

333

Hg2+ standard solution into the pretreated water samples. The recoveries of Hg2+ in

334

these fortified samples were obtained in range of 87%-125%, which further indicating

335

the immunoassay is a reliable and satisfactory method for detecting Hg2+ in real

336

samples.

337

Table 1. Recoveries tests of Hg2+ in the spiked real water samples (n = 3) Sample

Added

Found

RSD

Recovery

(ng mL−1)

(ng mL−1)

(%)

(%)

Tap water 1

1

1.11±0.06

5.8

111

Tap water 2

5

4.45±0.17

3.8

89

Tap water 3

10

9.06±0.22

2.4

91

River water 1

1

1.25±0.09

7.5

125

River water 2

5

4.40±0.16

3.7

88

River water 3

10

9.11±0.30

3.2

91

Lake water 1

1

1.03±0.07

6.6

103

Lake water 2

5

4.73±0.16

3.4

95

Lake water 3

10

8.65±0.26

3.0

87

338

ACKNOWLEDGMENT We gratefully acknowledge the Science Fund from the National Natural Science Foundation of China (No. 21075087, No.21175097), the Project of Scientific and Technologic Infrastructure of Suzhou (SZS201207), and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.


Page 19 of 35


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Page 25 of 35


340

Table 1. Recoveries tests of Hg2+ in the spiked real water samples (n = 3) Sample

Added

Found

RSD

Recovery

(ng mL−1)

(ng mL−1)

(%)

(%)

Tap water 1

1

1.11±0.06

5.8

111

Tap water 2

5

4.45±0.17

3.8

89

Tap water 3

10

9.06±0.22

2.4

91

River water 1

1

1.25±0.09

7.5

125

River water 2

5

4.40±0.16

3.7

88

River water 3

10

9.11±0.30

3.2

91

Lake water 1

1

1.03±0.07

6.6

103

Lake water 2

5

4.73±0.16

3.4

95

Lake water 3

10

8.65±0.26

3.0

87

341 342



Page 26 of 35

343

Figure captions

344

Scheme 1. Illustrative ECL detection mechanism for Hg2+ based on GCE/PDDA-GN-

345

CdSe/chitosan/Ag/Ab-AuNPs-HRP.

346

Figure 1. (A) HRTEM image of PDDA-GN, (B) HRTEM image of PDDA -GN-CdSe

347

composite, (C) HRTEM image of CdSe QDs and (D) UV–vis absorption spectra: (a)

348

CdSe QDs, (b) PDDA-GN-CdSe composite and (c) PDDA-GN.

349

Figure 2. UV–vis absorption spectrum (A) and HRTEM (B) of AuNPs.

350

Figure 3. EIS of (a) bare glassy carbon electrode, (b) GCE/PDDA-GN-CdSe, (c)

351

GCE/CdSe, (d) GCE/PDDA-GN-CdSe/chitosan, (e) GCE/PDDA-GN-CdSe/chitosan

352

/Ag and (f) GCE/PDDA-GN-CdSe/chitosan/Ag/BSA in 0.1 mol L−1 KCl solution

353

containing 5 mmol L−1 [Fe (CN)6]3−/4−.

354

Figure 4. ECL curve and cyclic voltammogram (inset) of the detection Hg2+ at 10 ng

355

mL−1 in oxygen-saturated pH 9.0 Tris–HCl buffer containing 0.1 mM KNO3 and 1.5

356

mM OPD at 100 mV s−1.

357

Figure 5. Optimizations of concentrations of (A) coating antigen and (B) antibody,

358

and (C) volumes of 1mg mL−1 HRP for immunoreactions and enzymic catalytic

359

reaction.

360

Figure 6. Cyclic ECL curves of GCE/PDDA-GN-CdSe/chitosan/Ag/Ab-AuNPs-HRP

361

for Hg2+ detection at (a) 1000 ng mL−1, (b) 100 ng mL−1, (c) 10 ng mL−1, (d) 5 ng

362

mL−1, (e) 1 ng mL−1, (f) 0.5 ng mL−1 in oxygen-saturated pH 9.0 tris-HCl buffer

363

containing 0.1 M KNO3 and 1.5 mM OPD. Inset: linear calibration curve for Hg2+

364

detection (n = 3).

365

Figure 7. (A) Selectivity of the developed immunosensor for Hg2+ detection over

366

other metal ions. The concentrations of the metal ions are 100 ng mL-1. (B)

367

Continuous cyclic scans of immunosensor formed at 0.5 ng mL−1, 1 ng mL−1, 10 ng

368

mL−1, 100 ng mL−1 and 500 ng mL−1 Hg2+ standard solutions, respectively. Other

369

conditions are the same as in Figure 4.


Page 27 of 35


370

TOC Art

371

372



Scheme 1. Illustrative ECL detection mechanism for Hg2+ based on GCE/PDDA-GN- CdSe/chitosan/Ag/AbAuNPs-HRP. 157x92mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 28 of 35

Page 29 of 35


Figure 1. (A) HRTEM image of PDDA-GN, (B) HRTEM image of PDDA -GN-CdSe composite, (C) HRTEM image of CdSe QDs and (D) UV–vis absorption spectra: (a) CdSe QDs, (b) PDDA-GN-CdSe composite and (c) PDDA-GN. 147x135mm (300 x 300 DPI)



Figure 2. UV–vis absorption spectrum (A) and HRTEM (B) of AuNPs. 81x41mm (300 x 300 DPI)


Page 30 of 35

Page 31 of 35


Figure 3. EIS of (a) bare glassy carbon electrode, (b) GCE/PDDA-GN-CdSe, (c) GCE/CdSe, (d) GCE/PDDAGN-CdSe/chitosan, (e) GCE/PDDA-GN-CdSe/chitosan /Ag and (f) GCE/PDDA-GN-CdSe/chitosan/Ag/BSA in 0.1 mol L−1 KCl solution containing 5 mmol L−1 [Fe (CN)6]3−/4−. 85x76mm (300 x 300 DPI)



Figure 4. ECL curve and cyclic voltammogram (inset) of the detection Hg2+ at 10 ng mL−1 in oxygensaturated pH 9.0 Tris–HCl buffer containing 0.1 mM KNO3 and 1.5 mM OPD at 100 mV s−1. 74x65mm (600 x 600 DPI)


Page 32 of 35

Page 33 of 35


Figure 5. Optimizations of concentrations of (A) coating antigen and (B) antibody, and (C) volumes of 1mg mL−1 HRP for immunoreactions and enzymic catalytic reaction. 55x18mm (600 x 600 DPI)



Figure 6. Cyclic ECL curves of GCE/PDDA-GN-CdSe/chitosan/Ag/Ab-AuNPs-HRP for Hg2+ detection at (a) 1000 ng mL−1, (b) 100 ng mL−1, (c) 10 ng mL−1, (d) 5 ng mL−1, (e) 1 ng mL−1, (f) 0.5 ng mL−1 in oxygen-saturated pH 9.0 tris-HCl buffer containing 0.1 M KNO3 and 1.5 mM OPD. Inset: linear calibration curve for Hg2+ detection (n = 3). 85x72mm (300 x 300 DPI)


Page 34 of 35

Page 35 of 35


Figure 7. (A) Selectivity of the developed immunosensor for Hg2+ detection over other metal ions. The concentrations of the metal ions are 100 ng mL-1. (B) Continuous cyclic scans of immunosensor formed at 0.5 ng mL−1, 1 ng mL−1, 10ng mL−1, 100 ng mL−1 and 500 ng mL−1 Hg2+ standard solutions, respectively. Other conditions are the same as in Figure 4. 63x25mm (300 x 300 DPI)


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