Plasmonic ELISA Based on Nanospherical Brush-Induced Signal

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A Plasmonic ELISA Based on Nanospherical Brush-Induced Signal Amplification for the Ultrasensitive Naked-eye Simultaneous Detection of the Typical Tetrabromobisphenol A Derivative and Byproduct Zhen Zhang, Nuanfei Zhu, Shuaibing Dong, Menglu Huang, Liuqing Yang, Xiangyang Wu, Zhenjiang Liu, Jiahao Jiang, and Yanmin Zou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02803 • Publication Date (Web): 01 Aug 2017 Downloaded from http://pubs.acs.org on August 2, 2017

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Journal of Agricultural and Food Chemistry

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A Plasmonic ELISA Based on Nanospherical Brush-Induced Signal Amplification for the Ultrasensitive Naked-eye Simultaneous Detection of the Typical Tetrabromobisphenol A Derivative and Byproduct

6 7 8

Zhen Zhang1*, Nuanfei Zhu1, Shuaibing Dong1, Menglu Huang1, Liuqing Yang3,

9

Xiangyang Wu1*, Zhenjiang Liu1, Jiahao Jiang1, Yanmin Zou2

10 11

1

12

212013, China

13

2

School of Pharmacy, Jiangsu University, Zhenjiang 212013, China

14

3

School of Chemistry & Chemical Engineering, Jiangsu University, Zhenjiang 212013,

15

China

School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang

16 17 18 19

* Corresponding author:

20

E-mail: [email protected], [email protected]

21

Fax:

+86-511-88790955

22

ACS Paragon Plus Environment


23

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Abstract

24

Based on H2O2-mediated growth of gold nanoparticle (AuNPs), a novel

25

plasmonic enzyme-linked immunosorbent assay (pELISA) was developed with a

26

polyclonal antibody for the ultrasensitive simultaneous naked-eye detection of

27

tetrabromobisphenol

28

tetrabromobisphenol A mono(hydroxyethyl) ether (TBBPA MHEE), one of the major

29

derivatives and byproducts of tetrabromobisphenol A (TBBPA), respectively. In this

30

modified indirect competitive pELISA, glucose oxidase (GOx) played an important

31

role leading to the growth of AuNPs through a reaction between GOx and glucose to

32

produce hydrogen peroxide (H2O2). In addition, further signal amplification was

33

achieved via a large number of GOx molecules, which were immobilized on silica

34

nanoparticles carrying poly brushes (SiO2@PAA) to increase the enzyme load, and

35

the whole complex was conjugated on the second antibody. Under the optimized

36

conditions, 10-3 µg/L TBBPA DHEE can be distinguished via the observation of a

37

colored solution, and the limit of detection (LOD) of the method using a microplate

38

reader reaches 3.3×10-4 µg/L. In contrast, the sensitivity of the method was 3 orders of

39

magnitude higher than that using conventional colorimetric ELISA with the same

40

antibody. Furthermore, the proposed approach showed good repeatability and

41

reliability after a recovery test fortified with a variety of targets was performed

42

(recoveries, 78.00% - 102.79%; coefficient of variation (CV), 4.38% - 9.87%). To our

43

knowledge, this is the first case in which pELISA was applied for the detection of

44

small molecules via the production of H2O2 from GOx and glucose. The method will

A

bis(2-hydroxyetyl)

ether

(TBBPA


DHEE)

and

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45

be widely used for the investigation of TBBPA DHEE and TBBPA MHEE in real

46

environments.

47

Keywords:

48

Immunoassay, Silica Nanoparticles, Glucose Oxidase, Tetrabromobisphenol A,

49

Derivative, Byproduct

Plasmonic

Enzyme-linked

Immunosorbent

50


Assay

(pELISA),


51 52

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Introduction Tetrabromobisphenol

A bis(2-hydroxyetyl)

ether

(TBBPA DHEE)

and

53

tetrabromobisphenol A mono(hydroxyethyl) ether (TBBPA MHEE) were considered

54

as a typical derivative and byproduct of tetrabromobisphenol A (TBBPA, the most

55

extensively used brominated flame retardant), respectively1, 2, which were produced

56

both intentionally and unintentionally along with TBBPA. Some studies indicated that

57

the chemicals have similar characteristics as persistent organic pollutants, such as

58

environmental persistency and bioaccumulation3, and other research showed that the

59

pollutants were neurotoxic4. Considering that the extensive use of the chemicals could

60

result in their widespread occurrence in the environment, it is necessary to develop a

61

simple and reliable method to investigate the pollutant levels in the environment and

62

to perform a risk assessment.

63

In fact, an analytical approach has been established for the detection of TBBPA

64

MHEE using ultra-high performance liquid chromatography-Orbitrap Fusion Tribrid

65

mass spectrometry2. However, the method required extremely expensive instruments

66

and sophisticated sample pretreatment; these drawbacks limited the application of the

67

method for the analysis of a large number of environmental samples5. Although Tian

68

et al. reported a method involving derivatization with AgNO3 for TBBPA DHEE6, 7,

69

this method is difficult to be applied for real sample measurements because it can

70

damage the mass spectrometer. To solve the above-mentioned problems, we produced

71

a polyclonal antibody capable of recognizing TBBPA DHEE and TBBPA MHEE, and

72

based on this antibody, a simple, high-throughput indirect competitive enzyme-linked


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73

immunosorbent assay (ELISA) was established with a limit of detection (LOD) of

74

0.7018

75

Information). However, the sensitivity of the method did not meet the requirement for

76

trace

77

high-throughput immunoassays are needed.

µg/L (DOI:10.1016/j.envpol.2017.05.076,

pollutants

analysis

in

the

environment.

shown

in

Therefore,

the

more

Supporting

sensitive

78

The colorimetric assay with gold nanoparticles (AuNPs) aggregation/growth

79

garnered our attention because it could be utilized as a signal reporter for sensitive

80

naked-eye chemical and biomolecular detection without using sophisticated

81

instruments8-17, especially in the plasmonic ELISA (pELISA) application, which is

82

simple, high-throughput and sensitive12,

83

hydrogen peroxide (H2O2) is a key factor to control the accumulation of gold

84

nanoparticles. When a large amount of H2O2 exists, gold ions will quickly be reduced

85

to spherical or regular gold nanoparticles, and the reaction solution would turn red;

86

conversely, less H2O2 would bring about irregular AuNPs with a blued solution. Based

87

on which, Stevens et al. developed a sensitive sandwich pELISA that depends on the

88

catalase (CAT)-induced AuNPs growth for the determination of prostate-specific

89

antigen (PSA) and HIV-1 capsid antigen p24, whose limit of detection (LOD) was

90

10-18 g/mL21. To increase the sensitivity of traditional pELISA, Huang, X. et al.

91

introduced a nanospherical brush (SiO2@PAA) into the analytical system to decrease

92

the affinity of competing antigens and increase the catalase loading as a CAT

93

container22-24. Consequently, the LOD of the above study was 7 orders of magnitude

94

higher than that using conventional pELISA22. All of these were accomplished via

18-21

. In the assay, the concentration of



95

H2O2 consumption to obtain the signals. In contrast, some research indicated that

96

glucose oxidase (GOx) was employed to produce H2O2 for colorimetric

97

measurement16, 25; hence, we speculated that the GOx-glucose system could be used in

98

pELISA as a new method to change the H2O2 concentration.

99

In this study, a novel improved indirect competitive pELISA was designed for

100

the ultrasensitive detection of TBBPA DHEE and TBBPA MHEE, and our method

101

was performed by producing H2O2 in the pELISA system instead of consuming H2O2,

102

as reported in other approaches. In the assay, the kinetic growth of AuNPs was

103

controlled by an interaction between GOx and glucose as a method of regulating the

104

H2O2 concentration. Furthermore, to improve the sensitivity of the indirect

105

competitive pELISA, SiO2@PAA was introduced into the assay as a label with GOx

106

for signal amplification, which was grafted onto the second antibody (Ab2).

107

Experimental section

108

Reagents and materials

109

Gelatin, Na2HPO4·12H2O, NaH2PO4·2H2O, KH2PO4, NaCl, KCl, CaCl2,

110

Na2CO3, and NaHCO3 were obtained from Sinopharm Chemical Reagent Co. Ltd

111

(Shanghai, China). Tween-20 was purchased from Sigma-Aldrich (USA), and

112

2-(N-morpholino) ethanesulfonic acid (MES), HAuCl4·3H2O, N-hydroxysuccinimide

113

(NHS), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), GOx,

114

and β-D-glucose were purchased from Aladdin (USA). Goat Anti-Rabbit IgG

115

antibody was purchased from Sigma-Aldrich (USA). The polyclonal antibodies

116

against TBBPA DHEE and TBBPA MHEE were produced in our lab, and detailed


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information is provided in the Supporting Information.

118

Buffers and solutions: A series of glucose concentrations was prepared by

119

diluting a stock solution in 1 mM 2-(N-morpholino) ethanesulfonic acid buffer (MES).

120

A carbonate buffer solution (CBS) composed of 1.59 g Na2CO3 and 2.94 g NaHCO3

121

was dissolved in 1.0 L pure water and stored at 4oC in a refrigerator. Blocking buffer

122

was obtained by adding 1% gelatin in the CBS solution. The washing buffer was

123

phosphate-buffered saline (PBS), which contained 0.01 mol/L phosphate and 0.05%

124

Tween-20 at pH 7.4.

125

The 96-well microplates were was purchased from NUNC (Denmark). All the

126

chemicals were of analytical grade and used without further purification. The protein

127

solutions were stored at 4°C before use. A UV−vis spectrophotometer (Model:

128

UV-2600, SHIMADZU, Japan), a transmission electron microscope (TEM) (Model:

129

JEM-2100, JEOL, Japan), and a microplate reader (Model: Infinite M1000 Pro,

130

TECAN, Switzerland) were used for the analyses.

131

Method feasibility evaluation

132

In brief, different concentrations of glucose and 0.5 U/mL GOx were added into

133

the microplate with 100 µL/well and 50 µL/well, respectively. After incubating the

134

plates for 1 h at 50 °C, 50 µL freshly prepared 0.2 mM gold (III) chloride trihydrate in

135

1 mM MES buffer was mixed with the solution. When the control reactions acquired a

136

red tonality, the nanoparticle growth process was stopped by adding 50 µL 100 mM

137

glutathione to each well. The absorbance was measured using a UV−vis

138

spectrophotometer. The results are provided in the Supporting Information (Figure.



139

S7).

140

Immobilization of GOx on SiO2@PAA

141

SiO2@PAA was kindly provided by Professor Xiong and Xu, and the

142

nanomaterial could be synthesized according to previously reported studies22, 26, 27.

143

GOx was covalently immobilized on SiO2@PAA with an efficient “chemical

144

conjugation after electrostatic entrapment” (CCEE) method28, and the procedure was

145

described as following: 2 mg of SiO2@PAA was suspended in 1.0 mL of 50 mM MES

146

buffer (pH = 5.0) and mixed with 4 mg GOx at room temperature for 4 h. After

147

removing the excess GOx through centrifugation and washing with PBS three times,

148

the electrostatic absorption of GOx was converted into a covalent bond with the

149

addition of 0.5 mL 50 mM EDC and 0.5 mL 10 mM NHS. The conjugation was

150

allowed to proceed at room temperature for 24 h. After centrifuging the solution and

151

discarding the supernatant, the obtained SiO2@PAA@GOx conjugates were washed

152

with PBS buffer and stored in this solution at 4°C at a concentration of 1 mg/mL.

153

Preparation of SiO2@PAA@GOx@Ab2

154

The SiO2@PAA@ GOx@Ab2 complexes were prepared through the reaction of

155

the carboxyl and amide groups between Ab2 and SiO2@PAA@GOx; this reaction was

156

performed using a classical carbodiimide (EDPC) method29-32. In short, 1 mg

157

SiO2@PAA@GOx resuspended in 1 mM PBS buffer was reacted with 1 mL 50 mM

158

EDC and 1 mL 10 mM NHS under stiring overnight at room temperature. After this

159

step, Ab2 was added into the mixture, and the reaction proceeded for 24 h. Then, the

160

obtained Ab2 complex was purified using protein dialysis membranes with PBS buffer


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for 72 h and then stored in 1 mL PBS buffer at 4°C for further use.

162

Development of the indirect competitive pELISA

163

The polystyrene microtiter plates were coated with coating antigen (TBBPA

164

DHEE-OVA, diluted in CBS buffer) overnight at 4°C; then, the strips were blocked

165

with blocking buffer for 2 h at 37°C after they were washed 3 times with washing

166

buffer. Afterwards, 50 µL TBBPA-DHEE antibody and 50 µL TBBPA-DHEE were

167

added into each well. Following a 30 min incubation at 37 °C, the microtiter plates

168

were washed with washing buffer to remove the unbound compounds, 100 µL 0.5

169

µg/mL SiO2@PAA@ GOx@ Ab2 was added into every well, and the reaction

170

proceeded for 30 min at 37°C. Next, with the addition of 100 µL glucose (10 mM),

171

the solution was incubated for 45 min at 50°C. After the washing step, 50 µL freshly

172

prepared 0.2 mM gold(III) chloride trihydrate in 1 mM MES buffer was mixed with

173

the solution. When the control reactions acquired a red tonality, the nanoparticle

174

growth process was stopped by adding 50 µL 100 mM glutathione to each well. The

175

absorbance at 550 nm was measured using a microplate reader. For comparison, the

176

indirect competitive ELISAs based on horseradish peroxidase (HRP) were conducted,

177

and the procedures for HRP-based ELISA are provided in the Supporting Information

178

(Figure S6).

179

Results and discussion

180

Design strategy

181

Scheme 1 depicts the assay protocol of the novel indirect two-step pELISA for

182

the simultaneous detection of TBBPA-DHEE and TBBPA-MHEE. The pELISA was



183

designed based on the fact that GOx could catalyze the oxidation of glucose to

184

generate H2O2. The produced H2O2 could stimulate the growth of AuNPs with the

185

addition of AuCl4- 9, 16, as a result, the reaction solution would turn red or blue in

186

response to the amount of H2O2. Hence, the common indirect pELISA was developed

187

for determination of targets when GOx was conjugated with Ab2; however, the

188

method suffered from insufficient sensitivity in the application of trace pollutants

189

analysis. Considering that nanospherical brushes (SiO2@PAA) possess the

190

advantages26,

191

abundant carboxyl groups in its molecular structure that are beneficial for linking with

192

the amino groups of enzymes23; large surface area, which is suitable for increasing the

193

enzyme load and decreasing the affinity of competing antigens. Therefore, in the assay,

194

Ab2 was labeled with SiO2@PAA@GOx, and the H2O2 concentration was regulated

195

by the GOx amount. If the sample solution contains a low concentration of targets,

196

then after competition with coating antigens, more Ab2 conjugated with

197

SiO2@PAA@GOx would be captured by the antibodies against the targets in the

198

microplate wells, leading to more H2O2 production. Large amounts of H2O2 would

199

catalyze the HAuCl4 solution to form regular spherical AuNPs with the addition of

200

MES, and the solution would generate a red color. In contrast, a high target

201

concentration would result in a blue-colored solution because of producing irregular

202

AuNPs.

203

Conjugation and characterization of SiO2@PAA@GOx

204

28, 33

, such as good biocompatibility, which was attributed to the

In the assay, the conjugation of SiO2@PAA and GOx was considered as an


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205

important step for signal amplification, and several experimental factors that could

206

affect the conjugation performance were investigated in detail, including the pH of the

207

buffer and EDC, NHS, and GOx concentrations. Meanwhile, the capacity and activity

208

of the immobilized GOx were identified as important parameters to evaluate this

209

relationship.

210

SiO2@PAA modification by GOx was accomplished using CCEE28, and the first

211

step of the approach could be achieved by adjusting the buffer pH value to that of the

212

isoelectric point of the protein. In the present study, MES buffers with varied pH

213

values were tested to obtain the optimum conditions, as shown in Figure 1A. The

214

capacity of immobilized GOx increased substantially to the maximum value when the

215

pH reached 5; then, a decreasing tendency was observed when the pH exceeded 5,

216

which was in agreement with the isoelectric point of GOx (pH = 4.9). Hence, the

217

MES buffer at pH 5 was selected for this reaction system. The next step was to

218

achieve a strong bond between the enzyme and the nanosilica spheres via the amino

219

groups on the surface of GOx and the carboxyl groups of the SiO2@PAA by the active

220

ester method. Figure 1C and Figure 1B indicate that the capacity of immobilized

221

GOx reached its maximum when the EDC and NHS concentrations were 50 mM and

222

10 mM, respectively. Furthermore, during the enzyme coupling process, the addition

223

of NHS had no obvious side effects on the enzyme activity. In addition, when the

224

GOx concentration was 4 mg/ml, the best capacity and activity of immobilized GOx

225

were achieved (Figure 1D).

226

A series of further verifications were performed for SiO2@PAA@GOx, and its



227

catalytic properties were evaluated. The TEM image shows that SiO2@PAA was

228

monodispersed with a core-shell structure (Figure 2D), and successful immobilization

229

of GOx on SiO2@PAA can be intuitively observed in Figure 2A (the solution of e

230

turned from light yellow to colorless after centrifugation compared with the color in a

231

and c ). In addition, the UV-visible absorption spectra in Figure 2C provides powerful

232

evidence for our results; the characteristic absorption of SiO2@PAA@GOx was 449

233

nm, which was different from that of free GOx and SiO2@PAA but similar to that

234

previously reported34. To investigate the potential effects on the catalytic performance

235

after the covalent conjugation between GOx and SiO2@PAA was achieved, the

236

solutions (containing HAuCl4 and glucose) were visually evaluated in the presence or

237

absence of our synthesized SiO2@PAA@GOx. Figure 2B indicates that the color

238

changed only for the solutions in the presence of SiO2@PAA@GOx and a decrease in

239

the SiO2@PAA@GOx concentration (diluted gradually) caused the colored solution

240

to turn from red to blue, which implied that SiO2@PAA@GOx had sufficient catalytic

241

ability and could be used in the following pELISA.

242

Optimization of the indirect competitive pELISA

243

In this assay, the H2O2 concentration was considered as the key factor influencing

244

the pELISA performance, which was related to several factors, including the glucose

245

concentration, the concentration of SiO2@PAA@GOx-labeled Ab2 and the reaction

246

time between glucose and the labeled Ab2. To control the amount of produced H2O2,

247

these three parameters were optimized.

248

Glucose was used as a type of reaction substrate to control the production rate


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249

and yield of H2O2; as shown in Figure 3A, the solutions with 0.1 mmol and 1 mmol

250

H2O2 displayed a blue and light red color, respectively, indicating that the production

251

of H2O2 is too low to meet our requirements. By comparison, the 10 mmol glucose

252

solution displayed a pure red color, and there was a clear distinction between the red

253

and blue colors, which was suitable for our pELISA. The concentration of

254

SiO2@PAA@GOx-labeled Ab2 was the second element affecting the quantity of

255

produced H2O2. Figure 3B shows that more sensitivity was observed when the

256

concentration of SiO2@PAA@GOx was 0.5 µg/mL. Theoretically, when the reaction

257

is allowed to proceed for a longer period, more H2O2 is generated, and accordingly,

258

the solution turns dark red. Considering the colors and the detection limit of this

259

method, we selected 45 min as a suitable reaction time (see Figure 3C).

260

Under the above-mentioned optimized conditions (glucose, 10 mmol;

261

SiO2@PAA@GOx-labeled Ab2, 0.5 µg/mL; reaction time, 45 min), the sensitivity of

262

the proposed approach was estimated by the naked eye and the microplate reader via

263

measurement of serial TBBPA-DHEE concentrations. As shown in Figure 4A,

264

obvious color changes were observed after different target concentrations were added

265

(from 10-4 µg/L to 103 µg/L), and these color changes could be clearly discriminated

266

visually at the TBBPA-DHEE concentration of 10-3 µg/L to 103 µg/L. In addition, the

267

sensitivity

268

concentration-dependent absorbance changes of the analytes. Figure 4B reveals that

269

there was a good linear relationship with the concentration of analytes from 10-3 µg/L

270

to 10 µg/L, and the regression equation for TBBPA-DHEE was y = 0.37532-0.02536

was

measured

by

a

microplate

reader


according

to

the


271

Lg(x) with a good correlation coefficient (R2 = 0.98914). The LOD was calculated

272

(based on a signal/noise ratio (S/N) of 3) as 3.3×10-4 µg/L, which was 3 orders of

273

magnitude lower than that of the conventional indirect competitive ELISA (0.7018

274

µg/L) using the same antibody.

275

Assay validation and measurement of real samples

276

The accuracy and precision of the proposed pELISA were evaluated using a

277

spike-recovery analysis for water samples from three sources fortified with different

278

concentrations of targets. Satisfactory recoveries (78.0% - 102.79%) were obtained

279

after sample analysis with four replicates, and the intra-assay coefficient of variation

280

(CV) ranged from 4.38% to 9.87%.

281

To further investigate the reliability of the established method, it was applied to

282

measure real samples along with conventional ELISA. Table 2 indicates that the

283

results using both methods were in good agreement at a high level for targets.

284

Meanwhile, our pELISA showed a higher sensitivity, and ELISA could not meet the

285

requirement for the determination of trace analytes.

286

In the present study, an improved indirect competitive pELISA was developed

287

for the ultrasensitive simultaneous detection of TBBPA-MHEE and TBBPA-DHEE

288

using our produced antibody, and SiO2@PAA served as a GOx container for signal

289

amplification. Under the optimized conditions, the LOD of the proposed method

290

reached 10-3 µg/L using the naked eye and 3.3×10-4 µg/L using a microplate reader.

291

Due to the satisfactory accuracy and precision of the established approach (recoveries

292

of 78.0% - 102.79%; CV values of 4.38% - 9.87%), it was applied for the analysis of


Page 14 of 30

Page 15 of 30


293

real samples along with conventional ELISA, and the results of the two methods were

294

agreed well. Therefore, the method has a great potential for application to the

295

determination of trace pollutants in environmental and food samples.

296

Acknowledgements

297

The present work was supported by the National Natural Science Foundation of

298

China (Grants 21577051, 41601552), the Natural Science Foundation of Jiangsu

299

Province (BK20140543), and the Jiangsu Collaborative Innovation Center of

300

Technology and Material of Water Treatment. In addition, we appreciate the professor

301

Yonghua Xiong in Nanchang University and professor Hong Xu in Shanghai Jiao

302

Tong University for giving us SiO2@PAA, and professor Xiong also provided some

303

assistances for us in the synthesis of SiO2@PAA.

304

Supporting Information Available

305

Additional information as noted in text. This material is available free of charge

306

via the Internet at htpp://pubs.acs.org.

307

Notes

308

The authors declare no conflict of interest.



Page 16 of 30

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423 424



425

Page 22 of 30

TABLES Table 1. The recovery of the indirect competitive pELISA (n=4)

426

Background

Added

Found

Recovery

CV

(µg/L)

(µg/L)

(µg/L)

(%)

(%)

0.0100

0.0091

91.00

8.22

0.0500

0.0416

83.20

5.13

0.5000

0.4332

86.64

5.99

5.0000

5.1396

102.79

4.38

0.0100

0.0083

83.00

7.56

0.0500

0.0391

78.20

9.87

0.5000

0.4011

80.22

7.19

5.0000

4.8226

96.45

6.01

0.0100

0.0101

101.0

7.99

0.0500

0.0392

78.40

8.91

0.5000

0.4117

82.34

8.72

5.0000

4.7819

95.64

7.24

Sample

Pure Water

Tap Water

River water

ND

ND

ND

427

ND: Not be detected; CV, intra-assay coefficient of variation obtained from 4 determinations

428

performed in the same plate.

429 430 431 432


Page 23 of 30


433

434

Table 2. The Detection of real water samples in Suzhou (n=4) Samples

ELISA (µg/L)

Our Work (µg/L)

S1

ND

ND

S2

ND

0.32

S3

ND

ND

S4

7.7

6.9

S5

7.2

6.9

S6

2.6

3.0

S7

1.7

1.8

S8

1.5

1.4

S9

ND

0.091

S10

ND

ND

S11

ND

ND

S12

ND

0.11

ND: Not be detected

435 436 437 438 439 440 441 442 443 444



445

SCHEMES

446 447

Scheme 1. Schematic illustration of indirect competitive pELISA（Ab1: primary

448

antibody;

Ab2: secondary antibody）

449


Page 24 of 30

Page 25 of 30


450

FIGURE CAPTIONS

451

Figure 1. The optimization of conjugation parameters (A. The pH of buffer; B. The

452

concentration of EDC; C. The concentration of NHS; D. The concentration of GOx).

453

Figure 2. The characterization of synthetic compounds (A. Images of aqueous

454

suspensions of SiO2@PAA before and after immobilizing with GOx, (a): free GOx, (b)

455

and (c): dispersed SiO2@PAA and SiO2@PAA@GOx, (d) and (e): centrifugated

456

SiO2@PAA

457

SiO2@PAA@GOx, (a): not add in, (b): add in; C. The UV/vis spectra of aqueous

458

suspensions of free GOx, SiO2@PAA and SiO2@PAA@GOx; D. The TEM image of

459

SiO2@PAA).

460

Figure 3. The parameter optimization of indirect competitive pELISA (A. The

461

concentration of glucose; B. The concentration of SiO2@PAA@GOx@Ab2; C. The

462

time for the reaction between GOx and glucose).

463

Figure 4. Indirect competitive pELISA dectection for TBBPA-DHEE (A. Naked-eye

464

detection of TBBPA-DHEE; B. Calibration curves toward TBBPA-DHEE).

and

SiO2@PAA@GOx;

B.

Validation

465


for

the

catalysis

of


466 467

Figure 1. The optimization of conjugation parameters (A. The pH of buffer; B. The

468

concentration of EDC; C. The concentration of NHS; D. The concentration of GOx).

469


Page 26 of 30

Page 27 of 30


470 471

Figure 2. The characterization of synthetic compounds (A. Images of aqueous

472

suspensions of SiO2@PAA before and after immobilizing with GOx, (a): free GOx, (b)

473

and (c): dispersed SiO2@PAA and SiO2@PAA@GOx, (d) and (e): centrifugated

474

SiO2@PAA

475

SiO2@PAA@GOx, (a): not add in, (b): add in; C. The UV/vis spectra of aqueous

476

suspensions of free GOx, SiO2@PAA and SiO2@PAA@GOx; D. The TEM image of

477

SiO2@PAA).

and

SiO2@PAA@GOx;

B.

Validation

478


for

the

catalysis

of


479 480

Figure 3. The parameter optimization of indirect competitive pELISA (A. The

481

concentration of glucose; B. The concentration of SiO2@PAA@GOx@Ab2; C. The

482

time for the reaction between GOx and glucose).

483


Page 28 of 30

Page 29 of 30


484 485

Figure 4. Indirect competitive pELISA dectection for TBBPA-DHEE (A. Naked-eye

486

detection of TBBPA-DHEE; B. Calibration curves toward TBBPA-DHEE). Each

487

point represents the mean values ± standard deviation for three replicates.

488 489 490 491 492 493



494

For TOC only

495


Page 30 of 30

Plasmonic ELISA Based on Nanospherical Brush-Induced Signal

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