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A Highly Selective and Sensitive Electrochemical Immunoassay of Cry1C Using Nanobody and #-# Stacked Graphene Oxide/Thionine Assembly Qing Zhou, Guanghui Li, Yuanjian Zhang, Min Zhu, Yakun Wan, and Yanfei Shen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02945 • Publication Date (Web): 12 Sep 2016 Downloaded from http://pubs.acs.org on September 13, 2016

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Analytical Chemistry

1 2

A

3

Immunoassay of Cry1C Using Nanobody and π-π π Stacked

4

Graphene Oxide/Thionine Assembly

5 6 7 8

Qing Zhoua†, Guanghui Lib†, Yuanjian Zhanga, Min Zhub, Yakun Wanb,* and Yanfei Shena,*

9

a

Highly

Selective

and

Sensitive

Electrochemical

School of Chemistry and Chemical Engineering, Medical School, Southeast

10

University, Nanjing 210009, China

11

b

12

Chinese Academy of Sciences, Shanghai 201203, P.R. China.

13

Email: [email protected] (Y. Shen); [email protected] (Y. Wan).

14

† Q. Z. and G. L. contributed equivalently.

CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,

15

1

ACS Paragon Plus Environment


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16

Abstract

17

Cry1C is one of the emerging toxin proteins produced by the Bacillus thuringiensis in

18

the genetically modified crops for the pest control in agriculture, thus it is vital to

19

measure the Cry1C level in crops for the health and environmental concerns. Current

20

detections of Cry1C mainly rely on instrumental analysis such as high-performance

21

liquid chromatography, which are time-consuming and are generally cost-prohibitive.

22

Herein, a simple nanobodies (Nbs)-based electrochemical immunosensor has been

23

firstly proposed for highly selective and sensitively detection of Cry1C. The Nbs pair,

24

i.e., Nb51 and Nb54, which bind to different epitopes on Cry1C, were screened out

25

from an immunized Bactrian camel, with extra benefit of higher stability compared

26

with conventional antibodies. Further by using a π-π stacked graphene oxide/thionine

27

assembly that had fast electron-transfer kinetics as an electroactive label, the

28

immunoreaction that occurred between the two Nbs and Cry1C can be highly

29

sensitively quantified by square wave voltammetry. The linear detection range was

30

from 0.01 ng·mL-1 to 100 ng·mL-1, and the low detection limit was 3.2 pg·mL-1. This

31

method was further successfully applied for sensing Cry 1C in spiked samples with

32

recoveries ranged from 100.17% to 106.69% and relative standard deviation less than

33

4.62%. This proposed assay would provide a simple highly sensitive and selective

34

approach for the Cry1C toxin detection, and be applicable to be extended to other

35

toxin proteins sensing in foods.

36

Keywords

37

Cry 1C, nanobody, electrochemical immunoassay, graphene oxide/thionine assembly 2


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38

Introduction

39

Bacillus thuringiensis (Bt) has been widely used in genetically modified (GM) crops

40

due to the ability of producing Cry proteins such as Cry1A for pest resistance.1-3

41

However, recent studies have shown that some insects have developed resistance to Bt

42

proteins. As a result, Cry1C protein was developed in response to insects resistance to

43

currently available Bt proteins such as Cry1Aa and Cry1Ab, since the toxin-mediated

44

insect resistance can be eliminated or postponed when Cry1C was combined with

45

Cry1A or other groups of Bt proteins.4,5 Although transgenic technology can

46

effectively depress the use of pesticides and improve the productivity and quality of

47

plants, the effect of GM plants on the long term human health and environmental risk

48

assessment is still controversial. Until now, different countries and regions issued

49

different GM organism labeling polices according to their national conditions.6

50

Therefore, in order to monitor the presence of GM crops, it is of great importance to

51

develop highly sensitive and specific strategy for the determination of Cry proteins in

52

foods.

53

Currently, there are a wide variety of detection methods available for Cry

54

proteins, including high-performance liquid chromatography (HPLC), mass

55

spectrometry (MS), polymerase chain reaction (PCR) and Western blotting.7,8 A

56

drawback of these methods is that they are time-consuming and are generally

57

cost-prohibitive. As a result, antibody-based immunoassay such as enzyme linked

58

immunosorbent assay (ELISA) using conventional antibodies came to be an

59

alternative way for the qualitative and quantitative determination of Cry proteins.9,10 3



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However, for the detecting of Cry1C, to our knowledge, only two publications are

61

available in the literature that reported the use of ELISA.11,12 Moreover, for both of

62

these two immunoassay methods, the instability of conventional antibody cannot be

63

avoided, greatly hindering their practical applications.

64

Recently, nanobody (Nb) or named variable domain of the heavy chain (VHH)

65

has been extensively investigated in fields such as drug exploitation.13,14 Compared

66

with traditional antibodies, Nb has unique physiochemical properties such as small

67

molecular mass (~ 15 kD), high affinity and specificity, sufficient solubility, high

68

thermal stability and acid-resistance, and lower consumption and immunogenicity.15

69

Moreover, the ease of functionalization of Nb with rational selected biomarkers such

70

as electroactive or fluorescent species, or biomolecules such as toxin proteins makes it

71

promising in task-specific sensing. Nevertheless, currently the application for Nb is

72

mostly confined in the field of drug exploitation, and only a few electrochemical

73

immunosensors using Nb as bioreceptor have been realized and explored.16-19

74

On the other hand, graphene oxide (GO) or that in a reduced form have been

75

widely used to improve the performance of biosensors by increasing loading of

76

probes,20-25 because of its unique electronic properties, large specific surface area and

77

biocompatibility.23-27 Moreover, the aromatic molecular structure of GO allows for a

78

noncovalent modification of functional units such as thionine (Th), an electrochemical

79

probe, while the carboxyl groups on GO not only ensure a good dispersibility in

80

aqueous solution but also facilitate a covalent linkage of other biomolecules such as

81

Nb.28,29 Herein, we developed a novel approach for selective determination of Cry1C 4


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82

by combination of a matched nanobody pair, namely, Nb51 and Nb54, and a π-π

83

stacked graphene oxide/thionine (GO-Th) hybrid-based probe with high sensitivity

84

and selectivity (Scheme 1). The corresponding immunosensor possessed a wide

85

calibration range of 0.01 ng·mL-1 to 100 ng·mL-1 and a low detection limit of 3.2

86

pg·mL-1, and was successfully applied to sensing Cry1C in real agriculture samples.

87

88 89

Scheme 1. Cry1C immunosensor fabrication process.

90 91

Experimental Section

92

Materials and Reagents

93

Cry1C and other toxins were purchased from YouLong Bio. Co., Ltd. (Shanghai,

94

China). Freund’s complete adjuvant, Freund’s incomplete adjuvant, anti-mouse

95

IgG-alkaline phosphatase, ampicillin and isopropyl β-D-1-thiogalactopyranoside

96

(IPTG) were purchased from Sigma-Aldrich (USA). Mouse anti-HA tag antibody and 5



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97

anti-mouse IgG were obtained from Covance (USA). Pst I, Not I, Nco I and BstE II

98

were obtained from NEB (USA). 96-well plate was purchased from Thermo Scientific

99

NUNC (Denmark). VCSM13 helper phages (Filamentous phage), TG1 cells, WK6

100

cells, plasmid pBAD and plasmid pBirA were obtained from Prof. Serge

101

Muyldermans’s lab (Laboratory of Cellular and Molecular Immunology, Vrije

102

Universiteit Brussel, Belgium). Graphene oxide (GO) was prepared by a modified

103

Hummers’

104

1-ethyl-3-(3-dimethylaminopropyl)

105

(NHS) were purchased from Sigma-Aldrich (Shanghai, China). Bovine serum

106

albumin (BSA) was purchased from Sunshine Biotechnology Co. Ltd. (Nanjing,

107

China). PBS solution was purchased from Sangon Biotech (Shanghai, China). Other

108

chemicals such as potassium chloride, potassium ferricyanide, and ethanol were from

109

Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). All reagents were of

110

analytical grade and used as received unless otherwise specified. Ultrapure water

111

(18.2 MΩ) was obtained from a Thermal Smart2 water purification system (USA).

112

Anti-Cry1C Nanobody Screening and Cross-reactivity Assay

method

from

natural

graphite.30

carbodiimide

Thionine (EDC),

acetate

(Th)

salt,

N-hydroxysuccinimide

113

In order to get an anti-Cry1C library with high quality, an immune phage library

114

specific to Cry1C was constructed. Firstly, a healthy Bactrian camel was immunized

115

with Cry1C-His antigen, which was of high purity and immunogenicity. All camel

116

experiments were conducted according to guidelines approved by the Institutional

117

Animal Care and Use Committee of Shanghai Institute of Materia Medica. After six

118

times’ immunization, the blood was collected, and total RNA of lymphocytes was 6


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119

extracted and transcribed to complementary deoxyribonucleic acid (cDNA). Then, the

120

VHH fragments were amplified by nested PCR followed by ligation to phagemid

121

pMECS. The phage display library was finally constructed by transforming the

122

ligation products into electro-competent TG1 Escherichia coli (E. coli) cells and 24

123

colonies were randomly selected to evaluate the insertion rate of the library.

124

The phage library was subjected to biopanning on 96-well microtiter plates as

125

described previously.31 Cry1C-His antigen was diluted with coating buffer (100 mM

126

NaHCO3, pH 8.2) to 100 µg·mL-1 and coated overnight at 4ºC (20 µg Cry1C-His per

127

well), with His protein in coating buffer as control. The plates were blocked using 200

128

µL 0.1% casein after being washed by PBS with 0.05% Tween-20 (PBST) for 2 h.

129

Then 100 µL of the phage library was added, incubated for 1 h, and unbound phage

130

was removed by washing with PBST for ten times. Bound phage was eluted with 100

131

µL of triethylamine (100 mM) for 10 min and immediately neutralized with 100 µL

132

Tris-HCl (1.0 M, pH 7.4). A 180 µL aliquot of the neutralized phage was used to

133

infect a 2 mL culture of log-phase E. coli TG1 cells for subsequent amplification. The

134

above processes represented one round of biopanning. After 2 rounds of biopanning,

135

the Cry1C-specific phages were enriched enough to perform the following research.

136

In total, 96 independent subtractive panning were randomly selected and tested by

137

periplasmic

138

Cry1C-specific VHHs were confirmed by sequencing of the positive colonies.

extraction

enzyme-linked

immnosorbent

assay

(PE-ELISA).

139

The recombinant phagemid of the identified positive colonies was extracted and

140

electroporated into E. coli WK6 cells, and grown in Terrific Broth containing 0.1% 7



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glucose, ampicillin (100 µg·mL-1), kanamycin (35 µg·mL-1) and 2 mM MgCl2. When

142

the culture reached an OD600 value of 0.6 to 0.9, 1 mM IPTG was added, followed by

143

shaking overnight at 28ºC. Protein was released by osmotic shock. Nanobodies (Nbs)

144

specific to Cry1C containing 6×His tag were purified with Ni-NTA metal affinity

145

chromatography according to the purification protocol. After eluting with PBS that

146

containing 500 mM imidazole, we assessed the purity and size of the Nbs using 15%

147

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed

148

by staining with Coomassie Brilliant Blue.

149

The specificity of Cry1C specific Nbs were measured by indirect ELISA. As the

150

Cry1C antigen contains a His-tag, different toxins were applied along with His-tag to

151

verify the specificity of selected Nbs. 100 µL of Cry1Ab, Cry1Ac, Cry1B, Cry1F and

152

FOLR1-His (folate receptor 1-His) containing a His-tag in 100 mM NaHCO3 were

153

coated onto microtiter plates with the concentration of 2 µg·mL-1. 100 µL of purified

154

Cry1C Nbs were added to characterize the specificity by using antibody mouse

155

anti-HA and anti-mouse IgG after blocking with BSA and washed with PBST. The

156

absorption was observed at 405 nm.

157

Fabrication of Nanobody-based Electrochemical Immunosensor

158

The GO-Th nanohybrid was prepared by ultrasonicating 10 mL of 0.1 M PBS

159

containing 10 mg GO and 1 mg of thionine for 1 h. Then, the supernatant was

160

collected by centrifugation at 3000 rpm for 20 min. Subsequently, 1 mL of the

161

as-obtained GO-Th dispersion was mixed with 100 µL of freshly prepared solution of

162

400 mM EDC and 200 mM NHS in 0.1 M PBS (pH 6), and the mixture was 8


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163

ultrasonicated at room temperature for 30 min. After that, 10 µL of 1 mg·mL-1 Nb54

164

in PBS was added to the above dispersion, and the solution was stirred at 4 oC for 12 h.

165

Finally, 10 mg of BSA was added to the solution and the solution was stirred for

166

another 1 h at room temperature. The resulting Nb54@GO-Th was stored at 4 oC

167

under darkness.

168

For the immunosensor fabrication, prior to surface modification, the glassy

169

carbon electrode (GCE, d = 3 mm) was polished with 1.0, 0.3 and 0.05 µm alumina

170

slurries, respectively, followed by successive sonication in ethanol and ultrapure water.

171

Then, GCE was electrochemically oxidized by cyclic voltammetry scanning from -1

172

V to 1 V in 0.5 M H2SO4 to form carboxylic acid groups on the surface. Afterwards,

173

10 µL of 0.1 M PBS solution (pH 6.0) containing 400 mM EDC and 200 mM NHS

174

was dropped onto GCE and incubated for 2 h. Then, GCE was rinsed with 0.1 M PBS

175

(pH 7.4) and 10 µL of 100 µg·mL-1 Nb51 in 0.1 M PBS was dropped onto the GCE

176

and incubated at 4 oC for 12 h. Following a rinse with buffer, the resulting Nb51

177

modified GCE was blocked by 1% (w/v) BSA in 0.1 M PBS for 0.5 h at 37 oC. After

178

rinsing, the electrode was incubated with Cry1C solution for 1 h at 37 oC, followed by

179

rinsing with PBS solution. Finally, 10 µL of Nb54@GO-Th solution was dropped

180

onto the modified GCE and incubated for 2 h at 37 oC. The Cry1C immunosensor was

181

finally obtained after the rinsing with PBS.

182

Measurement of Cry1C in Spiked Corn Samples

183

The spiked samples were prepared by spiking Cry 1C toxin into non-transgenic

184

corn samples. Briefly, 10 mL of the protein extraction solution (0.1 M PBS containing 9



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Page 10 of 27

185

0.1% BSA and 0.05% Tween-20) was added into one gram of the dried and

186

homogenized corn powder samples. After a gentle shaking at room temperature for 2

187

h, the suspensions were centrifuged at 10,000 g for 10 min. Then the supernatant was

188

diluted 1000-fold by 0.1 M PBS and spiked with Cry1C with four different

189

concentrations (0.1, 1, 10, 100 ng·mL-1). The mixed extracts were used for sample

190

analysis by proposed electrochemical immunoassay, and each spiked sample was

191

analyzed with three replicates. The final concentration of spiked samples was

192

determined by interpolation method according to the standard calibration plot.

193

Apparatus

194

Electrochemical experiments were performed on a CHI 600 workstation (CHI,

195

USA) at room temperature with a three-electrode system. The working electrode was

196

a modified glassy carbon electrode (GCE), the reference electrode was an Ag/AgCl (3

197

M KCl) and the counter electrode was a platinum wire. Square-wave voltammetry

198

(SWV) was performed in 0.1 M PBS (pH 7.4) with 4 mV potential steps, 25 Hz

199

frequency and 25 mV amplitude. Scanning electron microscopy (SEM) images were

200

obtained from a Phenom ProX scanning electron microscope (The Netherlands),

201

coupled with an X-ray energy dispersive spectrometer (EDS). UV−vis absorption

202

spectra were collected from a Cary100 UV−vis spectrophotometer (Agilent,

203

Singapore). Elemental analysis was performed on Elementar Vario MICRO

204

(Germany).

205 206

Results and Discussion 10


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207

Anti-Cry1C Nanobody Screening and Cross-reactivity Assay

208

To obtain VHH library anti-Cry1C with high affinity and specificity, a healthy

209

Bactrian camel was immunized with pure and immunogenic Cry1C-His. After six

210

times of immunization,32 the RNA of lymphocyte cells was extracted from the

211

collected blood and reversely transcribed to cDNA. Then the heavy chain antibody

212

variable region was amplified and the first PCR products were about 700 bp (Fig.

213

S1a). By taking the first PCR products as templates, about 400 bp fragments of the

214

second PCR were obtained (Fig. S1b). Afterwards, the VHH genes were ligated into

215

pMECS vector and transformed to TG1 E. coli cells, which were then cultured in

216

medium. The library was finally constructed after the estimation of library size and

217

insertion rate. The size of the library against Cry1C was estimated to be 1×109

218

colony-forming

219

sequence-difference could be obtained from the library. As shown in Fig. S1c, 24

220

individual colonies were randomly picked up to evaluate the correct insertion rate of

221

the library, which was about 95.8%, demonstrating the successful construction of a

222

VHHs library against Cry1C. Therefore, a high-quality anti-Cry1C immunized phage

223

library for the following biopanning was successfully obtained.

units

(CFU).

Thus,

the

Nbs

with

high

specificity

and

224

Phage display technique was applied for the selection of anti-Cry1C Nbs. Each

225

round of phage biopanning, ~ 2 × 109 phages and 2 × 1011 helper phages were used for

226

the amplification of phage particles which had binding affinity to Cry1C. As showed

227

in Fig. S1d and Fig. S1e, after two rounds of panning, the ratios increased from

228

3.6-fold in the first round biopanning to 1084-fold in the second one. Considering the 11



229

excellent diversity and large size of the phage library, the screening was halted and

230

positive colonies identification via PE-ELISA was performed. In this process, 96

231

randomly selected colonies from the two rounds of panning were subjected to

232

PE-ELISA, and the nucleotide sequences of positive clones were detected. Finally, the

233

amino acid sequences were deduced by multiple sequence alignment, and these VHHs

234

were accordingly classified into four families based on the diversity of amino acid

235

sequences in complementarity determining region (CDR)3 region (Fig. S1f). In the

236

following research, the anti-Cry1C Nbs were all among the four kinds of Nbs, namely

237

Nb2, Nb9, Nb51 and Nb54, respectively. (b) Absorbance at OD405 (nm)

(a)

2.0

Nb51 Nb54

1.5 1.0 0.5 0.0 Cry Cry Cry Cry Cry His Bla 1A 1A 1B 1C 1F -tag nk b c

(c)

(d)

0.8

0.4

0.0 238 239

1.2

Nb51 Nb54

Relative activity (%)

1.2 Relative activity (%)

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Page 12 of 27

0.5 1 2 3 Treated time at 37 oC (h)

Nb51 Nb54

0.8

0.4

0.0

0.5 1 2 3 Treated time at 70 oC (h)

Fig. 1. (a) Anti-Cry1C Nbs that were expressed and purified by NI-NTA superflow 12


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240

column and characterized by SDS-PAGE. (b) Cross-reactivity analysis based on

241

ELISA for anti-Cry1C Nbs identification. The thermal stability of Nb51 and Nb54 in

242

37 ºC (c) and 70 ºC (d) for 0.5 h, 1 h, 2 h and 3 h, respectively.

243

Four kinds of Nbs which had different amino acid sequences were subcloned into

244

the expression vector WK6 with an HA-tag and a His6-tag at C-terminal. Because of

245

the His6-tag, Nbs can be purified by NI-NTA affinity chromatography. After

246

ultrafiltration, SDS-PAGE analysis showed an event band at about 15 kD, which was

247

consistent with the theoretical value. The results demonstrated that the purity of Nbs

248

can reach more than 90% with yields of at least 5 mg·L-1 (Fig. 1a). In order to

249

obtained one pair of Nbs for the double-antibody sandwiched immunoassay, a match

250

pair analysis was conducted with the four Nbs by an epitope mapping method33. After

251

coupled with horseradish peroxidase (HRP), the four Nbs were characterized by

252

ELISA with the unmodified Nbs by adding tetramethylbenzidine (TMB) colored

253

liquid, and Nb51, Nb54 were validated bound to two different epitopes on Cry1C. As

254

a result, Nb51 and Nb54 were selected for the following characteristic verification

255

and immunosensor construction. As an example, Nb51 and Nb54 were used as a

256

capture and label antibody in this study, respectively. Nevertheless, it should be noted

257

that a similar performance of the immunosensor (including the amount of –NH2 group

258

for immobilization and labeling, and the interaction between Cry1C and each

259

nanobody) could be expected if the roles of Nb51 and Nb54 were exchanged,

260

ascribing to the followed reasons: (1) the amount of amino groups exposed on Nb51

261

and Nb54 for the immobilization and labeling are similar due to the similar amino 13



262

acid sequences and linear structure of Nb51 and Nb54;34 (2) Nb51 and Nb54 were

263

bound to two different single epitope on Cry1C.

264

In order to characterize the specificity of the as-obtained Nbs, cross-reactivity

265

analysis was performed with ELISA technique by choosing four kinds of toxins,

266

which are Cry1Ab, Cry1Ac, Cry1B and Cry1F. The results showed excellent

267

specificity of the Nbs (Nb51 and Nb54) towards Cry1C, and no cross-reactivity with

268

other toxins as well as His -tag (Fig. 1b).

269

Temperature has significant effects on the activity of antibodies, therefore the

270

thermal stability of Nbs was evaluated. The activity of Nbs was detected by ELISA

271

after incubating at 37ºC and 70ºC for 0.5 h, 1 h, 2 h and 3 h, respectively. As shown in

272

Fig. 1c and Fig. 1d, the activity (absorption at 405 nm) of Nbs, the Nbs kept at least

273

approximate 80% after the incubation, demonstrated high thermal stability of these

274

two Nbs. Therefore, the high thermal stability will make it possible for Nbs to be

275

applied in more applications such as clinical examinations and immunological

276

research, especially those requires high temperature, which are typically limited by

277

the low thermal stability of normal antibodies.

278

(a)

(b)

279 280

O C N S

64.60% 28.80% 4.70% 1.90%

GO-Th

(c) 0.8 Absorbance

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Page 14 of 27

GO

600 565

0.4

Th

611

0.0 O C

74.80% 25.20%

250

500 750 Wavelength (nm)

Fig. 2. SEM images of (a) GO-Th, (b) GO. Insets show the elemental atomic 14


Page 15 of 27

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281

percentage by EDS. (c) UV-vis absorption spectra of GO, and Th, and GO-Th.

282 283

Characterization of GO-Th Assembly.

284

GO is generally a good substrate candidate for the biosensor due to its large

285

surface area and unique electronic properties. In this sense, Th, an electrochemically

286

active probe, was non-covalently modified on GO via a π-π interaction, which was

287

confirmed by SEM images. Both the images of GO-Th (Fig. 2a) and GO (Fig. 2b)

288

demonstrated a similar wrinkled sheet-like structures, which was typically observed

289

for graphene-based materials, indicating that the intrinsic properties of GO were

290

preserved after assembly with Th. Moreover, the element analysis showed that

291

additional N and S elements were found in GO-Th with respect to GO, suggesting the

292

successful assembling Th on GO. In addition, based on the element analysis, the

293

average surface coverage for the Th molecules on GO surface was estimated to be

294

0.66×10-3 mol g-1(see more details in Supporting Information). The high average

295

surface coverage, attributed to the strong π-π interaction between GO and Th, and the

296

high surface area and water dispersibility of GO, would significantly improve the

297

sensitivity of the proposed immunosensor.

298

The successful assembly of GO and Th was further characterized by UV-vis

299

spectra. As shown in Fig. 3c, the original Th aqueous solution displayed two major

300

characteristic absorption peaks at ca. 280 nm and 600 nm, which were ascribed to the

301

π-π* transition of aromatic rings, and the n-π* transition of C=N bond, respectively.35

302

The main peak at 600 nm was characteristic of monomeric Th, while the 565 nm

303

shoulder can be attributed to the T-type dimer aggregate.36 The GO dispersion had a 15



304

maximum absorption at 230 nm and a shoulder around 290 nm, which were

305

corresponding to the π-π* transitions of aromatic C=C bonds and n-π* transition of

306

the C=O bond, respectively.37 For the GO-Th assembly, most absorption peaks of both

307

GO and Th were well retained, suggesting the successful assembly of GO and Th.

308

Nevertheless, it was noted that the 565 nm shoulder relatively increased compared to

309

the main peak at 600 nm, further confirming the aggregated state of Th on GO due to

310

the strong π-π interaction. More interestingly, a red shift up to 11 nm of the n-π*

311

transition of C=N bond for Th was observed, indicating an electron transfer between

312

GO and Th. Such effective electron-communication between GO and Th due to the

313

π-π interaction between them would greatly facilitate the electron transfer during the

314

electrochemical detection of Th probe in the proposed immunosensor.

-0.6

∆Ep = 110 mV

0 -6

-12 -0.6

Potential (V) -0.2 0.0

0.2

(b) 30

Current (µA)

6

GO-Th Th

-0.4

120

0 90 -30

∆Ep (mV)

(a) 12

Current (µA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 27

60

∆Ep = 50 mV -0.4

-0.2 0.0 Potential (V)

-60

0.2

0

300 600 900 Scan Rate (mV·s-1)

315 316

Fig. 3. (a) Cyclic voltammograms of GCE/GO-Th and GCE/Th in N2-saturated PBS

317

(0.1 M, pH 7.4), scan rate 100 mV·s-1. (b) Cyclic voltammograms of GCE/GO-Th in

318

N2-saturated PBS (0.1 M, pH 7.4) at scan rates of 10, 100, 200, 300, 400, 500, 600,

319

700, 800, 900, 1000 mV·s-1 (from inner to outer) and ∆Ep at different scan rates for 16


Page 17 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


320

GCE/GO-Th.

321 322

To further evaluate the effective electron transfer ability in the GO-Th assembly,

323

the cyclic voltammetry (CV) experiments at GO-Th modified GCE (GCE/GO-Th)

324

and Th modified GCE (GCE/Th) in N2-saturated PBS solution were performed,

325

respectively. As shown in Fig. 3a, a pair of redox wave was observed for both

326

GCE/GO-Th and GCE/Th, which could be ascribed to the redox waves of Th on the

327

electrode. Interestingly, the GCE/GO-Th displayed an anodic and cathodic peak

328

potential difference (∆Ep) of ~50 mV, which was only half of that of GCE/Th (~100

329

mV), implying that GO significantly improved the reversibility of the electrochemical

330

oxidation and reduction of Th at the electrode, most presumably by a strong π-π

331

interaction between GO and Th. More strikingly, the ∆Ep of GCE/GO-Th remained

332

nearly constant with the scan rate up to 1000 mV·s-1. Such fast electron

333

communication kinetics between the electrode and the electrochemical probe was

334

greatly anticipated for a highly sensitive electrochemical sensors.38

17



40

Current (µA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

20

Page 18 of 27

GCE/Nb51/BSA/Cry1C/Nb54@GO-Th GCE/Nb51/BSA/Cry1C GCE/Nb51/BSA GCE/Nb51 GCE

0 -20 -40 -0.2

335

0.0

0.2 0.4 Potential（ V）

0.6

336

Fig. 4. Cyclic voltammograms of modified GCE in 0.1 M PBS solution containing 2

337

mM K3[Fe(CN)6] during each step of the immunosensor construction, scan rate 100

338

mV·s-1.

339 340

Construction of the Electrochemical Immunosensor

341

The immunosensor configuration after each construction step was confirmed by

342

cyclic voltammetry (CV) measurements in 0.1 M PBS solution (pH 7.4) with 2 mM

343

K3[Fe(CN)6] as redox probe (Fig. 4).39 The CV of bare GCE showed a pair of

344

well-defined redox peaks with the anodic and cathodic peak potential difference less

345

than 85 mV, indicating a reversible electrochemical process. After Nb51, BSA and

346

Cry1C were successively modified on the surface of GCE, the redox peak currents

347

decreased gradually, which suggested that the electron transfer between K3[Fe(CN)6]

348

and GCE was blocked by the insulated biomolecules. Interestingly, the redox peak

349

currents are conversely enlarged when the Nb54@GO-Th probe was captured, which

350

could be ascribed to the enhanced electron transfer activity of GO in the assembly.40-42 18


(a) 4 Current (µA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


100 ng·mL-1 10 ng·mL-1

0.1 ng·mL-1 0.01 ng·mL-1

1 ng·mL-1

0.001 ng·mL-1 0 ng·mL-1

3

(b) 1.8

2 1 -0.6

1.6 Current (µA)

Page 19 of 27

∆I = 0.1665 lg C + 1.3965 R2 = 0.9967

1.4 1.2 1.0

-0.4

-0.2 0.0 Potential (V)

0.2

1E-3 0.01 0.1 1 10 100 Concentration (ng·mL-1)

351 352

Fig. 5. (a) The SWV curves and (b) the calibration curve of the immunosensor with

353

different concentrations of Cry1C toxin protein.

354 355

As shown in Scheme 1, after the sandwich immunoreaction, the Nb54@GO-Th

356

probe should be quantitatively captured via the formation of an immunocomplex.

357

Thus, the Nb54@GO-Th probe could be used to detect Cry1C sensitively and

358

quantitatively by SWV. It was found the SWV currents increased gradually with

359

increasing the Cry1C concentration (Fig. 5a). The calibration curve showed a good

360

linear relationship between the SWV current and logarithm values of Cry1C

361

concentrations in the range of 0.01-100 ng·mL-1 (Fig. 5b), indicating that the Cry1C

362

can be quantitatively detected by the proposed method. The linear regression equation

363

could be expressed as I (µA) = 0.1665 lg C (ng·mL-1) + 1.3965 with a correlation

364

coefficient of 0.9967. The limit of detection (LOD) was calculated to be 3.2 pg·mL-1,

365

using equation of LOD=3σ/S where σ is the standard deviation of blank samples

366

response and S is the slope of calibration curve. This LOD value was much lower than

367

the previous works using ELISA assay (see more detailed comparison in Table 1). 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 27

368

Moreover, the LOD was also much lower than that of the commercial kit of Cry1C

369

(such as Quantiplate Kit Cry1C in Leaf Tissue, USA, LOD = 0.2 ng·mL-1), and was

370

adequate to perform the determination in real samples.43 Therefore, the GO-Th could

371

be applied as an excellent redox label for the electrochemical sandwich immunoassay.

372 373

Table 1 Comparison of all current available methods for Cry1C detection a Methodb Nanobody based electrochemical immunosensor

Antibody

LOD

Nanobody (high 3.2 pg·mL-1 stable)

DAS-ELISAc

Mab (less stable)d

15 ng·mL-1

ic-ELISAe

ScFv (less stable)f

23 ng·mL-1

Features

References

High sensitive, high selective

This study

Less sensitive, less selective (simultaneous detection of 7 Cry1 toxins) Less sensitive, high selective

[11]

[12]

374

a

375

and are generally cost-prohibitive.7,8 b The assay time for the three methods in the

376

table are all about 3 h.

377

assay.

378

assay. f Single chain variable fragment.

Except for HPLC, MS, PCR and Western blotting that are time- and labor-intensive

d

c

Double antibody sandwich enzyme-linked immunosorbent

Monoclonal antibody.

e

Indirect competitive enzyme-linked immunosorbent

379 380

Specificity, reproducibility, stability and reliability

20


Page 21 of 27

1.6 1.2

Current(µA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.8 0.4 0.0

Cry1F Cry1B Cry1Aa Cry1C

381 382

Fig. 6. SWV peak currents of the immunosensor for Cry1F, Cry1B, Cry1Aa and

383

Cry1C with a concentration of 10 ng·mL-1 in PBS solution.

384 385

To investigate the specificity of the immunoassay, the modified electrodes

386

(GCE/Nb51/BSA) were incubated with Cry1F, Cry1B and Cry1Aa as interfering

387

toxin proteins. As shown in Fig. 6, the currents in Cry1F, Cry1B and Cry1Aa were

388

much lower than that of Cry1C, indicating that these interfering proteins could not

389

cause obvious signal variation and the proposed immunosensor possessed a good

390

selectivity. The reproducibility of the electrochemical immunosensor was evaluated

391

by six independent electrodes in the presence of 10 ng·mL-1 Cry 1C. The results

392

revealed that the biosensor showed good reproducibility with a relative standard

393

deviation

394

(GCE/Nb51/BSA/Cry1C/ Nb54@GO-Th) can keep 91.69% of initial response for 10

395

ng·mL-1 Cry1C after store in 0.1 M PBS of pH 7.4 at 4 oC for 15 days, indicating that

396

the proposed immunosensor had a satisfactory stability.

(RSD)

of as

low as

2.10%. In

addition,

the

immunosensor

21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

397

Page 22 of 27

Table 2 Recovery tests of Cry1C from spiked corn samples Corn sample

Spiked Cry1C

Found

Recovery (%)

RSD (%)

No.

(ng·mL-1)

1

0.1

0.1012

101.20

3.83

2

1

1.0669

106.69

2.70

3

10

10.2404

102.40

4.62

4

100

100.1699

100.17

2.31

398 399

To validate the reliability of the proposed electrochemical immunosensor, the

400

four different concentrations (0.1, 1, 10, 100 ng·mL-1) of the Cry1C toxin in

401

non-transgenic corn extract samples was measured. After triplicate measurements for

402

each sample, the concentration of Cry1C was determined by interpolation in the

403

standard calibration plot (Fig 5b), and the results were analyzed and listed in Table 2.

404

The recoveries of the spiked samples obtained ranged from 100.17% to 106.69%,

405

with a relative standard deviation less than 4.62%, which indicated that the

406

electrochemical assay had a good accuracy for quantitative detection of Cry1C toxin

407

based on Nbs in practical application.

408 409

Conclusion

410

In summary, a highly selective and sensitive electrochemical immunoassay was

411

firstly developed for Cry1C detection using Nb51 and Nb54 as the recognition units

412

and a π-π stacked GO-Th assembly as electrochemical probe. It was found that the 22


Page 23 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


413

Nbs were highly stable and selective for specific interaction with the target Cry1C

414

protein, and the GO-Th assembly not only provided abundant binding sites for the

415

immobilization of Nbs, but also had an excellent redox properties and high sensitivity

416

due to effective electron-communication via a non-covalent interaction. As a result,

417

the proposed immunosensor showed superior performance for Cry1C detection in

418

comparison with the current available assay methods with a remarkable selectivity,

419

excellent sensitivity, high reproducibility, a large linear concentration range and low

420

detection limit. The proposed sensor in this work would be promising to be applied to

421

Cry toxin proteins detection in many fields such as foods analysis, environmental

422

monitor and clinical diagnostics.

423 424

ASSOCIATED CONTENT

425

Supporting Information

426

The Supporting Information is available free of charge on the ACS Publications

427

website at DOI: 10.1021/acs.analchem.xxxx.

428

Figure S1: the Phage Library Construction and Anti-Cry1C Nbs identification, and the

429

details for the calculation of surface coverage of Th on GO.

430

AUTHOR INFORMATION

431

Corresponding Authors

432

*E-mail: [email protected] (Y. Shen);

433

*E-mail: [email protected] (Y. Wan).

434

Author Contributions 23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

435

†Q.Z. and G.L. contributed equally.

436

Notes

437

The authors declare no competing financial interest.

Page 24 of 27

438 439

Acknowledgements

440

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

441

(21305065, 21675022, 91333110, 31271365 and 31471216), Program from the

442

Natural Science Foundation of Jiangsu province (BK20130788 and BK20160028) and

443

the Fundamental Research Funds for the Central Universities. This work was also

444

supported by grants from Chinese Academy of Sciences (XDA12020332).

445 446

References

447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466

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