A novel immunochromatographic assay using ultramarine blue particles as visible label for quantitative detection of hepatitis B virus surface antigen

Journal Pre-proof A novel immunochromatographic assay using ultramarine blue particles as visible label for quantitative detection of hepatitis B virus surface antigen Jing Liu, Qiongqiong Yu, Guangying Zhao, Wenchao Dou PII:

S0003-2670(19)31394-7

DOI:

https://doi.org/10.1016/j.aca.2019.11.037

Reference:

ACA 237247

To appear in:

Analytica Chimica Acta

Received Date: 22 August 2019 Revised Date:

31 October 2019

Accepted Date: 13 November 2019

Please cite this article as: J. Liu, Q. Yu, G. Zhao, W. Dou, A novel immunochromatographic assay using ultramarine blue particles as visible label for quantitative detection of hepatitis B virus surface antigen, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.11.037. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier B.V. All rights reserved.

*Graphical Abstract

Graphical Abstract: Schematic illustration of preparation of antibody modified ultramarine blue particles and the principle of qualitative detection strategy of hepatitis B virus surface antigen

(HBsAg) with an immunochromatographic assay.

1

A novel immunochromatographic assay using ultramarine blue

2

particles as visible label for quantitative detection of hepatitis B

3

virus surface antigen

4

Jing Liu, Qiongqiong Yu, Guangying Zhao, Wenchao Dou

5 6

*1

Food Safety Key Laboratory of Zhejiang Province, School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310018, China

7 8

1 *

Corresponding author. Email address: [email protected] (W. Dou) 1

9

Abstract

10

Ultramarine blue particles as a novel visible label has been used to develop

11

immunochromatographic assay (ICA). The ultramarine blue particles, as a sodalite mineral

12

with formula: (Na,Ca)8[(S,Cl,SO4,OH)2(Al6Si6O24)], can generate a blue visible signal

13

were used as a label for ICA. Ultramarine blue particles were applied to a sandwich immunoassay

14

to detect hepatitis B virus surface antigen (HBsAg). Ultramarine blue particles were separated

15

from ultramarine blue industrial product by centrifugation. The polyacrylic acid (PAA) was used

16

to modify the carboxyl group on the surface of ultramarine blue particles. The goat anti-HBsAg

17

monoclonal antibody was modified on ultramarine blue particles by EDC/NHS activation of the

18

carboxyl groups. In the presence of HBsAg, the immune ultramarine blue particles were bound on

19

test line zone and forming a blue line on ICA strip which was directly readout by naked eye and

20

quantitatively measured by Image J software. Under optimal conditions, the color depth of test

21

line was linearly correlated with the concentration of HBsAg in concentration range from 1 to 50

22

ng mL-1. The calibration equation was y = 385.796 + 97.2298x (R2 = 0.9872), with limit of

23

detection (LOD) of 0.37 ng mL -1(S/N = 3). The sensitivity of this novel ICA was better than that

24

of ICA based on traditional gold nanoparticles as reporter probe. The ultramarine blue particles

25

offer an alternative type of visible label nanomaterial for the development of ICA.

26

Key words Immunochromatographic assay, HBsAg, Ultramarine blue particles, Visible,

27

Quantitative

28

2

29

Introduction

30

Immunochromatographic assay (ICA), also known as Lateral flow assay (LFA), is a rapid

31

analytical technique, with advantages such as simplicity, speediness, and sensitivity [1, 2]. ICA is

32

one of most successful and widely used point-of-care testing (POCT), and has been successfully

33

used in human chorionic gonadotropin (hCG) [3], serodiagnostic analyses [4, 5], cancer

34

detection[6, 7], cardiac markers[8] and infectious microorganisms detection[9]. Colloidal gold is as

35

the most extensively used label for ICA and shows good combination with antibody and strong

36

biocompatibility[10, 11]. However, colloidal gold based ICA su ers from low sensitivity because

37

of low bright intensity of colloidal gold[12]. For visible ICA, the color of test line is originated

38

from visible labels. In order to enhance the sensitivity of colorimetric ICA, the label nanomaterial

39

needs exhibit deep and bright color signal. It is an important strategy to enhance sensitivity by

40

exploiting new type color nanomaterial as labeling probes. Carbon nanotubes[13], bimetal

41

nanoparticles[14], colored silica nanoparticles [15, 16], etc. have been used as visible labels to

42

improve the high sensitivity ICA. However, the preparation and group function of nanoparticles are

43

always time-consuming, laborious and complicated. The complicated synthesis process results in

44

the poor repeatability of the final ICA strip. Therefore, there is still an urgent need to develop a

45

new type of cost-effective, easy-to-obtain and reproducible label to build novel ICA.

46

There are generally two classes of synthetic approaches for nanomaterial: top-down and

47

bottom-up methods. And it is still one scientific challenge to effectively and efficiently synthesize

48

nanomaterial by bottom-up method. The one of core challenge for bottom-up method is how to

49

produce nanomaterial in a large quantity at reasonable cost [17]. By contrast, top-down synthesis

50

strategy is one of the most efficient methods to synthesize nanomaterial [18]. 3

51

Industrial dyes were previously suggested for use in various analytical and diagnostic test

52

systems with visual assessment of results. Lubavina and coworkes developed a colloidal dyes

53

based competitive dot-immunoassay for low molecular substances[19]. Liu’ group using reactive

54

dyes and disperse dye as immunoassay markers of immunochromatographic test to detect human

55

serum albumin and antibodies against infectious bursal disease virus, respectively[20, 21]. Selahi

56

and Namavari et al developed a disperse dye immunoassay method for detection of antibodies

57

against Neospora caninum in cattle[22]. Carbon nanoparticles (CNPs) are also used as label of

58

signaling labels in rapid diagnostic assays as an alternative to gold and colored latex[23]. Industrial

59

dyes and CNPs are very cheap, stable and easy to prepare.

60

Ultrafine pigment is widely produced by top-down method, such as jet mill, boll mill, etc.,

61

with large quantity at reasonable cost. Ultramarine blue pigment, best known as artificial lapis

62

lazuli, is a non-toxic and environmentally friendly blue sodalite pigment and first synthesized by

63

Guimet at 1828 [24]. Ultramarine blue has very good properties[25]. It is an ideal pigment for paint,

64

ink, rubber, paper, printing and dyeing textile, plastics industry, culture and education, cosmetics,

65

construction and civil wall painting[26, 27]. Ultramarine blue can be industrially produced, and the

66

price is very cheap. However, the pigment including ultramarine blue has never been applied to the

67

field of visible analytical field.

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In this article, the ultramarine blue particles were first used as labels to build a quantitative

69

ICA strip for hepatitis B virus surface antigen (HBsAg) detection. The blue test line can be

70

observed simply with naked eyes or recoded using a camera and analyzed using Image J. This

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novel visible label has great potential to be applied in different analytical platforms for developing

4

72

different varieties of point-of-care tests. This ultramarine blue nanoparticle shows the potential to

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become a good visible probe for ICA platform to develop different kinds of ICA.

74

.

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Experimental Section

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Chemicals, materials and apparatus

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Ultramarine blue pigments were purchased from Longkou Haigerui Paint Co., Ltd. (Shandong,

78

China). Sodium hydroxide (NaOH) and polyacrylic acid (PAA) were received from Aladdin

79

Industrial Inc. (Shanghai, China). The conjugate pad, NC membrane, sample pad and absorbent

80

pad were bought from Weifang BND Biotechnology Co., Ltd. (Shandong, China). Deactivated

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hepatitis B virus surface antigen (HBsAg 3.5 mg mL-1), goat anti-mouse IgG, the mouse

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anti-HBsAg monoclonal antibody (anti-HBsAg mAb1, 1.7 mg mL-1) and capture (anti-HBsAg

83

mAb2, 8.2 mg mL-1) were purchased from Ebiocore Biotechnology Co., Ltd. (Hangzhou, China).

84

Fetal calf serum (FCS) was bought from Weifang BND Biotechnology Co., Ltd. (Shandong,

85

China). All others chemical reagents were of analytical grade. All the solutions were prepared with

86

ultra-pure water.

87

A Hitachi SU8010 scanning electron microscope (SEM) was obtained from Hitachi Inc.

88

(Tokyo, Japan); The Thermo Nicolet 380 Fourier transform infrared spectrometer (FTIR) was

89

provided by Thermo Fisher Scientific Co. (Shanghai, China); Malvern Nano 2S potential laser

90

particle

91

UK);Immunochromatography film device set purchased from Shanghai Jieyi Biotechnology Co.,

92

Ltd. (Shanghai, China).

93

Separation of ultramarine blue particles from ultramarine blue

analyzer

was

provided

by

Malvern

5

Instruments

Co.,

Ltd.(Worcestershire,

94

Ultramarine blue particles were prepared by sonication and centrifugation. 5 g of ultramarine

95

blue industrial product was weighed and dispersed in 50 mL of water to form a blue suspension.

96

The suspension was put in an ultrasonic cleaning machine for 10 min. The suspension was

97

centrifuged to collect deposits, washed with water. In this process, soluble impurity was removed

98

by centrifugation (10,000 rpm, 3 min). Thereafter, the deposits were dispersed in water. The

99

precipitation was collected by centrifugation cycles (1,000 rpm for 3 min) after the centrifugation,

100

the deposit was collected (ultramarine blue particles, <1,000 rpm). While the supernatant was

101

transferred to a fresh centrifugation tube and centrifuged at 4,000 rpm for 3 min, the deposit

102

(ultramarine blue particles, 1,000-4,000 rpm) and supernatant (ultramarine blue particles, >4,000

103

rpm) were separately collected. Finally, ultramarine blue particles having different particle sizes

104

can be obtained.

105

Preparation of the carboxyl functionalized ultramarine blue particles

106

45 mg of the above-mentioned selected ultramarine blue particles were dispersed in 10 mL of

107

water, 100 mg of PAA was added, and PAA was completely dissolved by ultrasonication. The pH

108

of the solution was adjusted to 8-9 with NaOH (1.0 M), and the reaction was sonicated for 1 h.

109

After the reaction was completed, it was washed three times with water. The final product was

110

stored in water for later use.

111

Preparation of antibody-modified ultramarine blue particles conjugates

112

Mouse anti-HBsAg monoclonal antibody (mAb1, 1.7 mg mL-1) was modified on the surface of

113

ultramarine blue particles, according to our previously reported method with some

114

modifications[28]. Briefly, 1 mg of ultramarine blue particles was dispersed in 1 mL of 0.05 M

115

MES (pH 6.0). 2 mg of EDC and 3 mg of NHS were added and sonicated for 15 min to activate 6

116

the carboxyl groups on the surface of the ultramarine blue particles. The activated particles were

117

washed twice with phosphate buffer (PB, 0.02 M, pH 7.4) to remove excess chemical reagent and

118

re-dispersed in 1 mL PB (0.02 M, pH 7.4). 14 µg of mAb1 was added and incubated with activated

119

ultramarine blue particles at room temperature for 2.5 hours, and 100 µL of mPEG-NH2 (0.1 g

120

mL-1) was added to neutralize the unreacted carboxyl group on the surface of the ultramarine blue

121

particles, and the reaction was continued for 1 hour. The final antibody modified ultramarine blue

122

particles were washed and re-dispersed in PB (0.02 M, pH 7.4) containing 1% BSA (w/v), 3%

123

sucrose (w/v) and 1% trehalose (w/v), 0.5% Tween-20. The concentration of final conjugates was

124

10 mg mL-1 and stored in a 4 °C refrigerator until use.

125

Preparation of ultramarine blue particles-based ICA strips

126

The composition of the ultramarine blue particles-based ICA system is shown in Fig. 1B. The

127

test strip consisted of four parts including nitrocellulose (NC) membrane, conjugation pad, sample

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pad and absorption pad. Firstly, the conjugation and sample pads were soaked in PBS (0.01M, pH

129

7.4) containing 0.5 % Tween-20, 1.0 % (w/v) BSA, 1.0 % (w/v) trehalose and 3.0 % sucrose (w/v)

130

and dried overnight at 37 °C. The anti-HBsAg mAb2 (1.5 mg mL-1) and goat anti-mouse IgG

131

antibodies (1 mg mL-1) were coated on to NC membranes as test (T) and control (C) lines at 1.0

132

µL cm-1 and then the NC membrane was dried at 37 °C for 2 h. Finally, the NC membrane,

133

conjugation pad, sample pad, and absorbent pad were sequentially adhered to the backing card, cut

134

into 4 mm width by strip cutter, which were stored at room temperature until use.

135

Measure procedure of ultramarine blue particles-based ICA

136

The HBsAg stock solution was diluted to a series of gradients (1-250 ng mL-1) with PBT (pH

137

7.4, 0.05% Tween-20) and PBT was used as a blank control. 80 µL of the test solution was added 7

138

dropwise to the sample pad. Qualitative results can be observed with the naked eye within 15 min.

139

For sensitivity detection, ultramarine blue particles-based immunochromatographic strips were

140

used to detect the sample solution. Each experiment was repeated at least three times. According

141

to previous reports[29], the quantitative measurements can be made by analyzing the gray value of

142

the test area using the camera combined with “ImageJ” software. The procedure of contrast

143

enhancing for negative images is described in Supporting Information.

144

Application in samples

145

In order to prove the practicability of the proposed ultramarine blue particles-based

146

immunochromatographic strip for real sample analysis. The sample solutions were prepared by

147

spiking HBsAg into fetal calf serum (FCS). Under the best optimized conditions, the detection

148

steps were as described above.

149

Results and Discussion

150

Characterization of ultramarine blue particles of different particle sizes and selection of the

151

right size

152

In this colorimetric ICA, the optical signal on test line is generated from ultramarine blue

153

particles. Therefore, the readout signal is largely related to the particle size and optical properties

154

of ultramarine blue particles (The UV-vis spectra of ultramarine blue particles as shown in Fig. 3C

155

with an absorption peak at 592 nm). Electron micrographs can clearly observe the morphology

156

and dispersion of ultramarine blue particles obtained at different centrifugal speed. As shown in

157

Fig. 2A, 2B and 2C, the shape of the ultramarine blue particles is irregular, and it can be seen that

158

the dispersion is very good. At the same time, the ultramarine blue particles obtained in this study

159

had good solution stability during storage (three months), and no flocculation or aggregation 8

160

occurred. As shown in Fig. 2D, The hydrodynamic diameter of ultramarine blue particles collected

161

at different rate (<1,000 rpm, 1,000-4,000 rpm, >4,000 rpm) are 809 nm, 384 nm, and 205 nm

162

respectively; the polydispersity indices (PDI) are 0.497, 0.314 and 0.071, respectively, indicating

163

that the isolated ultramarine blue particles (>4,000 rpm) has a good monodispersity and a narrow

164

size distribution.

165

In order to test whether the ultramarine blue particles can be used for ICA, the ultramarine blue

166

nanomaterials were added on the conjugated pad and flowed on the NC membrane of blank

167

immunochromatographic strip. The ultramarine blue nanomaterials of different particle sizes were

168

prepared into the same concentration (5 mg mL-1) and 5 µL of ultramarine blue particles solution

169

and 80 µL PB (pH 7.4, containing 0.05% Tween-20) were added dropwise to the conjugation pad.

170

After the desired running time, blank immunochromatographic strips were recorded by a digital

171

camera. As shown in inset of Fig. 2A and 2B, when the ultramarine blue particles (<1,000 rpm and

172

1,000-4,000 rpm) were used as visible label, a great amount of particles were aggregated at the

173

interface of conjugate pad and NC membrane. It is possible that the ultramarine blue particles

174

having a larger particle size are blocked and accumulate on the pores of the NC membrane,

175

therefore, the ultramarine blue particles (<1,000 rpm and 1,000-4,000 rpm) cannot be used for

176

ICA. However, as shown in inset of Fig. 2C, when the ultramarine blue particles (>4,000 rpm)

177

were added, there were no ultramarine blue particles detention and the NC membrane was very

178

clean. For the qualitative and quantitative ICA, the release of ultramarine blue particles and

179

background color are beneficial to improve the sensitivity and repeatability of ICA. In summary,

180

in this experiment, the ultramarine blue particles (>4,000 rpm) were selected to label antibody.

181

Characterization

of

carboxyl

functionalized 9

ultramarine

blue

particles

and

182

antibody-modified ultramarine blue particles conjugate

183

In order to achieve high performance for this ICA, the ultramarine blue particles need exhibit

184

strong biocompatibility and active group to ensure the binding of antibody on the ultramarine blue

185

particles. The FTIR spectra of ultramarine blue particles and carboxyl-modified ultramarine blue

186

particles are compared in Fig. 3A. The FTIR peaks at approximately 3466 cm−1 can be attributed

187

to O–H stretching vibrational mode. After the PAA coating, the peaks of the COO− asymmetric

188

stretching mode near 1637 cm−1 and COO− symmetric stretching mode near 1401 cm−1 of the

189

PAA carbonyl groups appeared[30]. These results suggest that successful modification of

190

ultramarine blue particles.

191

The hydrodynamic diameter of carboxyl functionalized ultramarine blue particles and

192

ultramarine blue particles-mAb1 conjugates were verified using a Malvern Nano ZS potential

193

laser particle analyzer. As shown in Fig. 3B, the size distribution curve of the ultramarine blue

194

particles-mAb1 conjugates slightly shifted to the right compared to the carboxyl functionalized

195

ultramarine blue particles, demonstrating an increase in particle size. According to the particle size

196

distribution curve, the average hydrodynamic diameter of the carboxyl functionalized ultramarine

197

blue particles was 225 nm, while the ultramarine blue particles-mAb1 conjugates average

198

hydrodynamic diameter was increased to 285 nm, and the hydrodynamic diameter corresponding

199

to the mAb1 was 22 nm. The Malvern Nano ZS potential laser particle analyzer show that in this

200

work, the ultramarine blue particles surface successfully modified the antibody.

201

Principle of the new ICA

202

The ultramarine blue is inorganic pigment with a well-known unique blue color. It is usually used

203

for painting coloring. The bright blue color of ultramarine blue provides new opportunity for the 10

204

application of ultramarine blue particles as a visible label for ICA. Fig. 1B illustrates the detection

205

principle of the established immunochromatographic strips. The sample solution (80 µL) was

206

added on sample pad, and then the liquid was carried along the membrane with the help of the

207

capillary attraction and the absorbent pad. The results were judged with naked eye after 15

208

minutes. If the sample solution contains HBsAg, the immunoprobe will combine with the subject

209

to form an ultramarine blue particles-mAb1-HBsAg complex. When the complex migrates to the

210

test zone, the test zone immobilizes the antibody capture complex, causing the composite

211

nanoprobe to accumulate in the test zone, forming a blue band visible to the naked eye. Excessive

212

immunoprobe will pass through the test line and bind to goat anti-mouse IgG at the control line

213

and form another visible blue line. When there is no HBsAg in the sample solution, only a blue

214

band of the control line can be observed. In all tests, the strips of the control line ensure the

215

validity of the immunochromatographic strip, otherwise the test is invalid.

216

The morphology of test zone in NC membranes is characterized by SEM before and after addition

217

of samples (Fig. 4). As shown in Fig. 4A, the NC film is a sponge-like three-dimensional structure

218

with a highly porous and rough surface with an asymmetric pore size of about 9 microns. As

219

shown in Fig. 4B, when there is no HBsAg in the sample solution, there is no agglomeration of

220

irregular particles in the NC channel because the nanoprobe and the immobilized mAb2 on the T

221

line do not form a sandwich structure. As shown in Fig. 4C, when there is HBsAg in the sample

222

solution, large amount of irregular ultramarine blue particles were adsorbed in the NC channel

223

because the nanoprobe-HBsAg binds to the immobilized mAb2 on the test line forming a

224

sandwich structure[31].

225

Optimization of the strip 11

226

The amount of anti-HBsAg mAb1 and labeling pH both affect the antibody activity and the

227

coupling efficiency in labeling process. The optimal amount of mAb1 and labeling pH were

228

determined by comparing the color depth of the T line on the strip after the addition of HBsAg, the

229

final gray value result is digitized by image J software. The object to be tested is HBsAg spiked at

230

50 ng mL-1, PBT as a buffer solution.

231

The amount of anti-HBsAg mAb1 on the ultramarine blue particles surface affects the

232

immunoreaction efficiency and sensitivity. The amount of mAbl in the conjugation solution was

233

first optimized. As shown in Fig. 5A, when different amount of antibody in range of 10–30µg is

234

applied, the highest value of the T line grayscale is achieved at 25µg. Thus the optimal amount of

235

anti-HBsAg mAb1 for immunoprobe preparation was 25µg.

236

The carboxyl functionalized ultramarine blue particles were incubated with anti-HBsAg mAb1 in

237

PB with different pH values. As shown in Fig. 5B, the gray value gradually increases with the

238

increase of the pH value, and the gray value reaches a maximum value at pH 7.5; when the pH

239

value exceeds pH 7.5, the gray value starts to decrease, pH 7.5 is the optimum coupling pH.

240

The band intensities are depended on the ultramarine blue particles-mAb1 conjugates captured on

241

the test line, which is corresponded to the amount of ultramarine blue particles-mAb1 conjugates

242

on the conjugate pad. To obtain maximum response using the minimal amount of ultramarine blue

243

particles-mAb1, the amount of ultramarine blue particles-mAb1 on the conjugate pad was

244

optimized by changing the volume of ultramarine blue particles-mAb1 loaded on the conjugate

245

pad. As shown in Fig. 5C that the gray value of the T line increase with an increase in volume of

246

the conjugate solution (1 to 4 µL). It can be seen that the gray value reaches a maximum when

247

volume of ultramarine blue particles-mAb1 is increased to 4 µL; further volume increase leads to a 12

248

decrease in the gray value. Therefore, 4 µL of ultramarine blue particles-mAb1 was used as the

249

optimal volume for the entire study.

250

Analytical performance

251

Under the optimized conditions, different concentrations of HBsAg (1-250 ng mL-1) were

252

detected by ultramarine blue particles-based ICA, PBT buffer was used as the control group, and

253

the results were recorded after reacted for 15 min. Each sample was examined in triplicate and

254

results are shown in Fig. 6A, it is can be seen that color intensity on the T-line increases with

255

increased HBsAg concentration. According to the definition, it can also be seen from Fig. 6A that

256

visual detection limit (VDL) for new strip is 1 ng mL-1.

257

Quantitative detection of HBsAg

258

The relationship between concentrations of HBsAg and the gray value (peak area) of test line

259

was further studied to realize the quantitative detection of HBsAg by ultramarine blue

260

particles-based ICA strips. As shown in Fig. 6B, the color depth of test line and the peak area of

261

gray value of test line have a good consistency, and they are both linearly increased with the

262

concentration of HBsAg. In the range of 1-50ng mL-1, the peak area of gray value of the test line

263

and the HBsAg concentration show a good linearity (Fig. 6D). The calibration equation is y =

264

385.796 + 97.2298x (R2 = 0.9872) and the detection limit (LOD) is 0.37 ng mL-1 (S/N = 3). We

265

compare our results with other nanomaterial-based ICA for qualitative and quantitative detection

266

of HBsAg (Table 1). Briefly, compared to Fe3O4 particles-based ICA and red SiNPs-based ICA,

267

the current method can greatly improve the sensitivity of HBsAg detection by 2-3 times.

268

Compared with the black Fe3O4 particles and colored silica spheres, the ultramarine blue particles

269

show deeper blue and result in a more sensitive detection for HBsAg test. The detection limit for 13

270

this work is higher than that of fluorescent microsphere-based ICA. Compared to this method, the

271

quantum dot method has higher sensitivity. However, the quantitative instruments used are

272

different, and immunochromatographic methods using fluorescence and quantum dot probes

273

require specialized reading instruments such as fluorescence spectrophotometers. There are also

274

some ultra-sensitive methods for detecting HBsAg. These methods require use of europium

275

chelate-loaded silica nanoparticles probes or hetero-assembled gold nanoparticle probes, and these

276

methods require special instrument quantification. Dual gold nanoparticle as probe that involves

277

biotin and streptavidin, which adds cost. This problem limits the use of fluorescent nanoparticles

278

in the development of portable ICA strip for on-site analysis. The result of ultramarine blue

279

particles-based ICA can be easily observed by naked eye without any special equipment. The

280

process is simple and has great advantages for quickly detecting proteins in poor areas.

281

Reproducibility and specificity of ultramarine blue particles-based ICA

282

The reproducibility of the ultramarine blue particles-based ICA strip was monitored by the ICA

283

strips of the same batch. During the reproducibility evaluation process, the gray intensities of the

284

test line was investigated at 4 levels with 0, 5, 20 and 50 ng mL-1 HBsAg, each sample was

285

assayed 6 times. The coefficient of variation are 5.81%, 7.15%, 4.18% and 5.39%, respectively,

286

suggesting the reproducibility of the ultramarine blue particles-based ICA strip is good.

287

To confirm the specificity of ultramarine blue particles-based ICA, three other antigens (AFP, CRP,

288

and HCV) and their complex mixtures (all in concentration of 1µg mL-1) with HBsAg was tested

289

by ultramarine blue particles-based ICA strips. As shown in Fig. 7, the ultramarine blue

290

particles-based ICA shows that all negative control signals were negligible compared to the

291

HBsAg sample. When the HBsAg is tested in the presence of other antigens, there is a slight 14

292

decrease in the signal value of the test line. It is confirmed that the ultramarine blue

293

particles-based ICA has good specificity and is slightly affected by the matrix.

294

Spiked serum sample analysis

295

In order to evaluate the detection ability of ultramarine blue particles-based ICA in serum samples,

296

FCS was used to simulate actual serum samples. The simulated actual sample test results are

297

shown in Fig. 8. Due to the influence of the matrix, the color of the test line is attenuated at

298

concentration of 1 ng mL-1, but at 5 ng mL-1 of HBsAg, there is still a blue detection line visible to

299

the naked eye, indicating ultramarine blue particles-based ICA can be used to detect HBsAg in

300

actual samples.

301

Conclusion

302

This study demonstrated the potential of using ultramarine blue particles labeled antibody as

303

the detector reagent to recognize the target in the ICA system. Ultramarine blue particles have

304

vivid blue color, good biocompatibility, good mono-dispersibility and low cost. And ultramarine

305

blue particles are very suitable for marking antibody in ICA. The sensitivity, specificity and spiked

306

sample experimental results proved that the ultramarine blue particles-based ICA strip is a

307

sensitive, reliable and economic immune tool for POCT detection of HBsAg in serum sample.

308

Moreover, this ultramarine blue particles-based ICA format is easily extended to other targets by

309

using corresponding antibodies.

310

Acknowledgment

311 312 313

Support by National Natural Science Foundation of Zhejiang Province (LY17C200003). Notes The authors declare no competing financial interest. 15

314

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18

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Figure caption

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Fig 1. (A) Surface Functionalization of ultramarine blue particles with Antibody against HBsAg;

426

(B) Schematic illustration of ICA detection of HBsAg.

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Fig 2: SEM photograph of ultramarine blue particles separated at different centrifugal speed:

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<1,000 rpm (A), 1,000-4,000 rpm (B), >4,000 rpm (C); inset: photographs of ICA strip after

429

addition of corresponding ultramarine blue particles;(D) Size distribution of ultramarine blue

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particles separated at different centrifugal speed, inset: photographs of carboxyl group modified

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ultramarine blue particles solution.

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Fig 3: (A) FTIR spectra of ultramarine blue particles (a), carboxyl modified ultramarine blue

433

particles (b); (B) Size distribution of carboxyl modified ultramarine blue particles(a) and antibody

434

modified ultramarine blue particles(b); (C) The UV-Vis spectra of the ultramarine blue particles.

435

Fig 4: SEM photographs of test zone NC membrane after spray of antibody (A) and after negative

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samples (B) and positive samples (C) flowing through ICA strip.

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Fig 5: The effect of amount of antibody used for modification of ultramarine blue particles (A),

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the pH (B); and volume of ultramarine blue particles-mAb1 on the ultramarine blue

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particles-based ICA (C).

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Fig 6: The color photographs (A) and contrast enhanced negative images (C) of the ultramarine

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blue particles-based ICA strips after addition different concentration of HBsAg samples. Standard

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calibration curve (B) and the linear response (D) for the quantitative analysis of HBsAg. Each data

443

point represents the average value obtained from three different measurements.

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Fig 7：Investigation of the ultramarine blue particles-based ICA specificity with the same

445

concentration of four proteins: HBsAg, AFP, CRP, HCV, and HBsAg solution in the presence of

19

446

antigens (AFP + CRP + HCV) at the concentration of 1 µg mL-1. FCS was used as a negative

447

control.

448

Fig 8: The color photographs (A) and contrast enhanced negative images (B) of the ultramarine

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blue particles-based ICA strips after detection of HBsAg in serum samples. The original HBsAg

450

solution was diluted with FCS to the following concentration gradient: 1, 5, 10, 50, 100, 250 ng

451

mL-1. Each sample was tested in parallel for three times using ultramarine blue particles-based

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ICA.

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Table 1: An overview on recently reported nanomaterial-based methods for visual determination

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of HBsAg

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20

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Fig 1

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21

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Fig 2

460 461 462 463

22

464

Fig 3

465 466

23

467

Fig 4

468 469

24

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Fig 5

471 25

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Fig 6

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26

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Fig 7

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Fig 8

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486 487

Table 1 Method

Label

detection limit

detection time

Reference

ICA

Fe3O4 particles

1 ng mL-1

-

[32]

20 min

[33]

0.075 ng mL

10 min

[4]

-1

10 min

[34]

-1

10 min

[35]

-1

ICA ICA ICA ICA

-1

Fluorescent microspheres

0.1 ng mL

-1

Quantum dot-beads Red silica nanoparticles

0.97 ng mL

Dual gold nanoparticle

0.06 ng mL

ICA

Europium chelate-loaded silica nanoparticles

0.03 ng mL

30 min

[36]

LSPR chip

Hetero-assembled nanoparticles

0.01 ng mL-1

10–15 min

[37]

ICA

Ultramarine blue particles

0.37 ng mL-1

15 min

This work

gold

488 489

.

29

Highlights


Ultramarine blue particles were separated from commercial ultramarine product.




Ultramarine blue particles were directly modified with antibody to use as visible label of immunochromatographic assay.




Ultramarine blue particles based immunochromatographic assay was established to detect

hepatitis B virus surface antigen (HBsAg).


This novel immunochromatographic assay was successfully used for sensitive detection of

HBsAg.

Conflict of interest

The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.