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Detection of multiple pathogens in serum using silicaencapsulated nanotags in a SERS-based immunoassay Jing Neng, Yina Li, Ashley J Driscoll, William C Wilson, and Patrick Alfred Johnson J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 7, 2018

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Journal of Agricultural and Food Chemistry

Detection of multiple pathogens in serum using silica-encapsulated nanotags in a SERS-based immunoassay Jing Nenga, Yina Lib, Ashley J. Driscollb, William C. Wilsonc, Patrick A. Johnsonb*

a

Current address: Department of Food Science and Technology，Zhejiang University of

Technology, Hangzhou, Zhejiang 310014, China. b

Department of Chemical Engineering, University of Wyoming, Laramie, WY 82071,

United States. c

Arthropod-Borne Animal Diseases Research Unit, Center for Grain and Animal Health

Research, USDA/ARS, Manhattan, KS 66502, United States.

*

Corresponding author at: Department of Chemical Engineering, University of

Wyoming, Laramie, WY 82071, USA, Tel.: +1 307 766 6524, E-mail address: [email protected] (P.A. Johnson).

ACS Paragon Plus Environment


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Abstract 1

A robust immunoassay based on surface enhanced Raman scattering (SERS) has been

2

developed to simultaneously detect trace quantities of multiple pathogenic antigens from

3

West Nile virus, Rift Valley Fever virus and Yersinia pestis in fetal bovine serum.

4

Antigens were detected by capture with silica-encapsulated nanotags and magnetic

5

nanoparticles conjugated with polyclonal antibodies. The magnetic pull-down resulted in

6

aggregation of the immune complexes and the silica-encapsulated nanotags provided

7

distinct spectra corresponding to each antigen captured. The limit of the detection was ~

8

10 pg/ml in 20% fetal bovine serum, a significant improvement over previous studies in

9

terms of sensitivity, level of multiplexing and medium complexity. This highly sensitive

10

multiplex immunoassay platform provides a promising method to detect various antigens

11

directly in crude serum samples without the tedious process of sample preparation, which

12

is desirable for on-site diagnostic testing and real-time disease monitoring.

13

Keywords

14

Surface-enhanced Raman scattering, multiplex immunoassay, magnetic nanoparticle,

15

West Nile Virus, Rift Valley Fever Virus, Yersinia pestis

16 17 18

INTRODUCTION

19

The development of surface enhanced Raman scattering spectroscopy (SERS) as a

20

detection modality for both portable diagnostic tools and high throughput laboratory


2

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1, 2

21

analysis has garnered significant attention over the past two decades

22

SERS include the large numbers of Raman compounds available as reporters for a wide

23

range of laser excitation wavelengths 3, the use of one single excitation wavelength for

24

multiple species

25

fingerprint-like signature profiles for each reporter. With these unique merits enabling

26

high level multiplex analysis 7-9, SERS has been successfully applied to develop complex

27

bioassays, such as multiple DNA detection

28

biomedical diagnosis

29

nanotags are less susceptible to photobleaching and self-quenching

30

laser at 785 nm used in this study can further circumvent background fluorescence issues

31

within biological matrices

32

application of the multiplex SERS-based immunoassays to highly complex biological

33

matrices, such as blood 21, embryos 22 and in vivo tumors 23.

34

To improve the stability of SERS nanotags and the specificity of multiplex SERS

35

bioassays, SERS nanotags with protective shells have been developed to prevent the

36

leaching of Raman reporters and capture biomolecules as well as to prevent cross-talk

37

between two different unshielded SERS nanotags which can lead to false positives 9.

38

Further, these SERS nanotags shield reporter molecules from reacting with contaminants

39

in the sample and prevent the nanotags from aggregating. Several approaches have been

40

used to fabricate the shell-protected SERS nanotags, such as silica encapsulation and

41

polymer coatings

42

biocompatibility, stability, optical transparency and ease of surface modification

43

two independent studies

4-6

24

. The merits of

and above all, the narrow Raman scattering peaks that confer

13-15

10, 11

and multiple protein detection

12

, for

and pathogen screening 16-18. Unlike fluorescent probes, SERS

20

.

19

. The near-infrared

Hence, the intrinsic robustness of SERS extends the

. Silica encapsulation has become widely adopted due to its

26, 27

25

. In

, gold nanoparticles were first incubated with Raman


3


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44

reporters before interacting with silane coupling agents, followed by the formation of a

45

silica shell via Stöber’s method

46

stabilize gold nanoparticles 29, however, competed with the adsorbed Raman reporters on

47

the gold surface. To address this issue, a layer-by-layer silica encapsulation approach was

48

developed, in which the addition of the polyacrylic acid layer eliminated the competitive

49

adsorption of thiol reagent, and thus improved the surface coverage of Raman reporters

50

20

51

bioassays in complex milieu 24, and the silica-encapsulated nanotags have been applied to

52

various biomedical diagnostics in biological matrices, ranging from detecting tumor cells

53

in whole human blood 21 to multiplexed imaging in living mice 30.

28

. The thiol-polyethylene glycol compound used to

. Overall, the protective shell on the nanotags improves the performance of SERS-based

54 55

The co-infection of multiple pathogens such as bacterial and viral pathogens in

56

agriculturally important animals and crops is not unusual in the same host

57

work, we report a robust and highly sensitive SERS-based immunoassay using magnetic

58

capture of silica-encapsulated SERS nanotags to simultaneously detect two viral antigens

59

and one bacterial antigen, West Nile virus (WNV) envelope (E) protein, Rift Valley fever

60

virus (RVFV) nucleocapsid (N) protein and capsular antigen fraction 1 (F1) from

61

Yersinia pestis in simulated serum samples with a portable Raman instrument. Early

62

detection of pathogenic antigens during infection has great significance in epidemic

63

prevention and therapeutic treatment. In the early stage of viral infection, the exogenous

64

antigen is generally of trace amount, which makes antigen detection within serum

65

challenging. To date, only a few studies have demonstrated simultaneous detection of two

66

or more pathogens in serum or blood with SERS-based technology. In a recent SERS-


31, 32

. In this

4

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67

based immunoassay study, femtogram detection limits of two antigens were achieved but

68

required hours of preparation

69

three antigens was recently reported as the first case of using the SERS-based lateral flow

70

assay with a Raman microscope system 34. In comparison, our SERS-based immunoassay

71

employing a colloidal particle system instead of the solid surface system, which

72

improved the reaction kinetics leading to a more efficient detection 35. To our knowledge,

73

this work is the first report of the use of a portable Raman instrument to quantitatively

74

and simultaneously detect three antigens of trace quantities in simulated serum samples in

75

under an hour with no sample preparation.

33

. In another advancement, the simultaneous detection of

76

77

MATERIALS AND METHODS

78

Reagents, Materials, and Basic Apparatus. Colloidal gold nanoparticles (60nm,

79

2.6×1010 particles/ml) were purchased from Ted Pella Inc. (Redding, CA) and Fe3O4

80

nanoparticles (~200 nm) were purchased from Nanostructured and Amorphous Materials,

81

Inc. (Houston, TX). Bis-N-succinimidyl-(pentaethylene glycol) ester linker (BS(PEG)12),

82

succinimidyl-[(N-maleimidopropionamido) dodecaethyleneglycol]) ester linker

83

(SM(PEG)12), ammonium hydroxide, Pierce™ protein A/G agarose and the Supersignal®

84

West Pico Chemiluminescent Substrate kit were obtained from Thermo Fisher Scientific

85

(Waltham, MA). HiTrap™ NHS-activated HP columns were ordered from GE

86

Healthcare (Piscataway, NJ). 3-aminopropyltrimethoxysilane (APTMS) (97%), tetraethyl

87

orthosilicate (TEOS) (98%), 3-mercaptopropyltriethoxysilane (MPTES), 3-

88

aminopropyltriethoxysilane (APTES), anhydrous dimethyl sulfoxide (DMSO), laser


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89

grade Nile blue A perchlorate (NB), Janus Green B (JG), methylene blue hydrate (MB)

90

and all other chemicals were from Sigma-Aldrich (St. Louis, MO). Chemical stock

91

solutions were made using deionized water (18.2 MΩ cm-1) prepared with Millipore Mill-

92

Q A10 purification system, which were filtered by passage through 0.2 µm Millipore

93

filtration units before use.

94

WNV E, RVFV N and F1 antigen/antibody(IgG) reagents. The recombinant His+-

95

tagged WNV E protein and rabbit anti-E polyclonal antibodies have been described

96

previously 36. Rabbit polyclonal antibodies raised against a recombinant His+-tagged

97

fusion of RVFV N protein lacking the transmembrane domain were prepared by Drolet et

98

al 37. Yersinia pestis capsular antigen fraction 1 (F1) and antibody were provided by Dr.

99

Gerard Andrews, Department of Veterinary Sciences, University of Wyoming.

100

Contaminating anti-E. coli antibodies were removed from serum by total E. coli protein-

101

bound HiTrap™ columns, and the polyclonal antibodies against F1 was then purified

102

from the serum by affinity chromatography with a Protein A/G resin. To evaluate

103

antibody/antigen recognition specificity, replicate Western blots containing three antigens

104

were probed individually with anti-E IgG, anti-N IgG and anti-F IgG, followed by

105

incubation with HRP (horseradish peroxidase)-conjugated goat anti-rabbit IgG and

106

chemiluminescence detection.

107

Synthesis of antibody-conjugated magnetic nanoparticles. The antibody-conjugated

108

magnetic nanoparticles were synthesized as previously reported 18 with slight

109

modification. First, 1 mg/ml of Fe3O4 nanoparticles in anhydrous DMSO were silanized

110

by dropwise addition of APTES to a final concentration of 5% (v/v) 38. The mixture was

111

shaken for 15 h at 40°C and an external magnet was then applied to collect Fe3O4


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nanoparticles, followed by several consecutive washings with DMSO to remove unbound

113

APTES. Afterwards, 2.5 mg of BS(PEG)12 linker was added to the amine-functionalized

114

Fe3O4 nanoparticles redispersed in 1 ml of DMSO. After 6 h incubation at room

115

temperature, the above nanoparticle/PEG conjugates were separated from solution by

116

magnetic collection and washed several times with phosphate buffered saline (PBS) at pH

117

7.5 to remove excess PEG linker and then resuspended in 1 ml PBS. Subsequently, three

118

40 µl aliquots of the nanoparticle/PEG conjugates were respectively reacted with 200 µl

119

of 0.2 mg/ml anti-E, anti-N and anti-F IgGs, followed by incubation for 6 h at room

120

temperature with gentle shaking, producing nanoparticle/PEG/anti-E,

121

nanoparticle/PEG/anti-N and nanoparticle/PEG/anti-F bioconjugates (referred to

122

hereafter as capture magnetic nanoparticles). Finally, the three bioconjugates were

123

washed repeatedly with PBS to remove unconjugated antibody and then resuspended in

124

240 µl of PBS for use in antigen-targeting detection assays.

125

Synthesis of silica-encapsulated and antibody-conjugated SERS nanotags. Silica

126

encapsulated SERS nanotags were synthesized by a layer-by-layer method 20. First, 20 µl

127

of appropriate concentration of Raman reporters (JG 10-3 M, NB 8 × 10-4 M, MB 2 × 10-4

128

M) was added into 6 ml of colloidal Au suspensions under vigorous stirring and the

129

mixture was equilibrated for 30 min. Next, 6 ml of polyacrylic acid (PAA) adjusted to pH

130

7.0 by 0.1 M NaOH was added dropwise into the mixture under rapid stirring for 3 h. The

131

PAA- encapsulated nanotags were washed three times to remove extra Raman reporters

132

and polymer molecules and then resuspended into water. APTMS was then added to a

133

final concentration of 3 × 10-7 M followed by a 30 minutes equilibration step. A silica

134

shell was subsequently grown by a modified Stöber’s method. Briefly, 17 ml of 2-


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135

propanol and 200 µl of ammonia hydroxide were added successively with gentle stirring.

136

Afterwards, 12 µl of TEOS was added in six portions over a time interval of 1 h and then

137

the mixture was reacted for 12 h. After addition of the TEOS, centrifugation (5000 rpm,

138

15 min) was applied to remove free silica nuclei. The collected silica-encapsulated

139

nanotags respectively labeled with JG, NB, and MB were resuspended in water and

140

adjusted to a neutral pH.

141

MPTES in ethanol was added to 600 µl aliquots of the silica-encapsulated nanotags to a

142

final concentration of 3 × 10-7 M. The mixture was incubated for 6 h at 37 ºC in a shaker.

143

After washing away unreacted MPTES by centrifugation, 0.25 mg of SM(PEG)12

144

dispersed in DMSO was added dropwise and the mixture was incubated for 6 h at room

145

temperature with gentle shaking. Following several washes with PBS to remove excess

146

linker and resuspension in PBS, the PEG-linked silica-encapsulated nanotags were

147

individually reacted with corresponding IgG-enriched antiserum (0.25 mg) for 3 h at

148

room temperature under gentle shaking. Janus Green labeled silica-encapsulated nanotags

149

were functionalized with anti-E IgG, NB-labeled silica-encapsulated nanotags with anti-

150

N IgG and MB-labeled silica-encapsulated nanotags with anti-F IgG. The antibody-

151

functionalized silica-encapsulated nanotags, referred to hereafter as reporter nanotags,

152

were washed several times by centrifugation/resuspension process and the final pellets

153

were resuspended in PBS at a desired concentration for multiplexing.

154

SERS detection of multiple antigen targets by magnetic capture. Multiplex assays

155

were conducted by mixing 45 µl of each reporter nanotag, 45 µl of 20% fetal bovine

156

serum containing dilutions of WNV E, RVFV N and F1 antigen, and 10 µl of each

157

capture magnetic nanoparticle. The ratio of each reporter nanotag and its corresponding


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158

capture magnetic nanoparticle was 1.1: 1. Triplicate reactions for each concentration of

159

antigen were incubated for 1 h at room temperature. The immune complexes were then

160

separated and concentrated from solution using a small external magnet (Ø20 mm × 10

161

mm). The concentrated pellets were interrogated by the laser of an Advantage NIRTM

162

Raman spectrometer (60mW, 785nm laser), (DeltaNu, Laramie, WY) fitted with a right

163

angle input optics accessory. Spectroscopic analysis was carried out using a custom

164

Python code in Jupyter Notebook. Raman spectra were recorded in a range of 200-2000

165

cm-1 with baseline off. Each recorded spectrum was an average of five independent data

166

acquisitions (1 s integration time).

167 168

Results and discussion

169

The TOC Figure illustrates the characteristics and mechanism of the immunoassay for

170

target antigen detection using capture magnetic nanoparticles and reporter nanotags.

171

Capture magnetic nanoparticles and reporter nanotags are incubated with a serum sample

172

containing target antigens. During incubation, the polyclonal antibodies conjugated on

173

magnetic nanoparticles and nanotags bind to different epitopes of the same antigen and

174

form immune complexes. These immune complexes are aggregated at the interrogating

175

laser spot by an external magnet source. For multiplex detection, immunoassays are

176

conducted by an assembly of the three capture magnetic nanoparticles and the three

177

corresponding reporter nanotags for WNV E, RVFV N and F1 antigens. The specificity is

178

based on the selection of antibody reagents that do not exhibit antigen cross recognition,

179

the choice of Raman dyes with distinguishable peaks that do not overlap with each other,


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and furthermore, the use of silica-encapsulated Raman nanotags that are impervious to

181

matrix and ionic effects, which is especially useful for detection in complex milieu such

182

as serum samples where heterogeneous proteins co-exist.

183

The thickness of silica shell plays an important role in the performance of silica-

184

encapsulated nanotags. A silica shell of ideal thickness should restrict massive

185

nanoparticle aggregation, minimize its interference in laser penetration at the same time,

186

and more importantly, it should depend on the diameter of the gold core 29. The thickness

187

of the silica shell synthesized on nanotags is approximately 10-15 nm (Figure 1. A),

188

which proved sufficient in blocking both the diffusion of Raman reporters away from the

189

surface and the penetration of external moieties while still maintaining high SERS

190

intensity. During the silica encapsulation, more than one nanotag can be encapsulated

191

together (Figure 1. B), and these clustered nanotags may enhance the Raman signal

192

intensity better than a single encapsulated nanotag 39.

193

One major difficulty in multiplex detection is the design of spectrally distinct SERS

194

nanotags with comparable intensities. When the intensities of the three reporters vary too

195

much, the signal of the strongest one can dominate the mixture, rendering it difficult to

196

distinguish and quantify the signal of weaker reporters. By adjusting the concentration of

197

each Raman dye in the silica shell, all three reporter nanotags were able to exhibit

198

distinguishable Raman signals with similar intensity and minimal overlap in the

199

immunoassay for multiplex detection, which is crucial for parallel detection of multiple

200

analytes (Figure 2).


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To evaluate multiplex assay performance in biological matrices, the antigens were spiked

202

in fetal bovine serum which was then diluted in PBS to a concentration of 20% (w/v)

203

with antigen concentration from 100 ng/ml to 10 pg/mL. The immunoassay yields intense

204

spectra, as shown in Figure 2. Spectrum A is from the multiplex immunoassay detecting

205

three antigens (10 ng/ml of each) in 20% fetal bovine serum and is characterized by

206

dominant peaks between 400-540 cm-1, 570-695 cm-1, and 1065-1160 cm-1, which are a

207

combination of three signals from methylene blue conjugated nanotags for F1 antigen

208

detection, Nile blue conjugated nanotags for RVFV N antigen detection, and Janus Green

209

labeled nanotags for WNV E antigen detection. Spectrum B is from an immunoassay

210

detecting only WNV E antigen (10 ng/ml), and displays only the characteristic peaks

211

between 1065-1160 cm-1, which are corresponding to the Janus Green labeled SERS

212

nanotags. Spectrum C is from an immunoassay detecting only RVFV N antigen (10

213

ng/ml), and the specific peaks between 570-695 cm-1 are consistent with the Nile blue

214

labeled SERS nanotags. Spectrum D is from an immunoassay detecting only F1 antigen

215

(10 ng/ml), and the distinctive peaks between 400-540 cm-1 match the methylene blue

216

labeled SERS nanotags. Spectra B, C and D confirm that the silica encapsulation prevents

217

cross-talk and increases the specificity of the multiplex immunoassay, for each of these

218

spectra shows only the characteristic peaks of one single reporter labeled SERS nanotag.

219

In the negative control immunoassay, all three antigens were omitted from reporter

220

nanotags and capture magnetic nanoparticles reagents only demonstrate negligible

221

background peaks (spectrum E).

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To determine the multiplex limit of detection for antigen capture, triplex spectra were

223

acquired for a broad range of F1, RVFV N and WNV E dilutions. As shown in Figure 3,


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a progressive decrease in intensity of characteristic peaks (between 400-540 cm-1, 570-

225

695 cm-1, and 1065-1160 cm-1) is observed as the concentration of the three antigens

226

decreased from 100 ng/ml to 10 pg/ml (A to F). The characteristic peaks in Spectrum F,

227

which were obtained from detecting 10 pg/ml of the three antigens, are still

228

distinguishable compared to Spectrum G, the negative control (Figure 3. inset).

229

Therefore, we conservatively conclude the limit of detection is a minimum of 10 pg/ml

230

(Spectrum F) in 20% fetal bovine serum.

231

To determine the quantitative response of SERS signal intensity to antigen concentration,

232

multiplex spectra were background corrected, and the integration areas under the

233

methylene blue peaks between 400-540 cm-1, Nile blue between 570-695 cm-1 and Janus

234

Green between 1065-1160 cm-1 were independently plotted as a function of their

235

respective antigen concentrations. A log-linear relationship for over three orders of

236

magnitude is found from 10 pg/ml to 10 ng/ml (Figure 4) and linear regression analysis

237

yields an R2 value of 0.9831 for F1 detection, 0.9424 for RVFV N detection and 0.9501

238

for WNV E antigen. The optimal concentration of Raman reporters and the best

239

stoichiometric ratio of reporter nanotags and capture magnetic nanoparticles are currently

240

under investigation.

241

We have demonstrated that a robust, high-level multiplex immunoassay based on SERS

242

can simultaneously detect three antigens in simulated serum samples by magnetic

243

aggregation

244

nanoparticles immune complexes. The limit of detection for WNV E, RVFV N and F1

245

antigen in 20% fetal bovine serum, 10 pg/ml, is 100-fold lower than the single antigen

246

capture assay in 10% serum

of

the

corresponding

40

silica-encapsulated

nanotags/antigen/magnetic

and the duplex antigen detection in PBS buffer


41

. In

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247

addition to the high sensitivity, this SERS-based immunoassay has high specificity and

248

robustness: the protective silica shell not only facilitates immobilization of the Raman

249

reporters on the SERS nanotag surface preventing cross-talk, but also blocks the

250

unfavorable competitive adsorption of the heterogeneous proteins in serum or blood

251

samples. Given that the sample size is of tens of microliters and it demands no

252

complicating operation such as sample preparation, this SERS-based immunoassay can

253

be applied to the onsite diagnostic testing and real-time disease monitoring of

254

agriculturally relevant animals. With little modification, this modality may be adapted for

255

the detection of a wide range of antigens in different biological matrices. Further

256

improvements will focus on expediting assay times, improving the long-term

257

performances of silica-encapsulated nanotags, and developing a portable Raman reader

258

that is specially engineered for magnetic capture of nanotags.

259

260

Acknowledgements

261

The authors thank the financial support from Kansas Bioscience Authority (KBA)

262

(Olathe, KS) and acknowledge the ABADR unit of CGAHR for providing recombinant

263

RVFV N antigens and rabbit anti-N polyclonal antibodies. We also appreciate receiving

264

F1 antigen and antibody reagents from Dr. Gerard Andrews (Department of Veterinary

265

Sciences, University of Wyoming). The contents of this publication are the responsibility

266

of the authors and do not necessarily represent the views of KBA and USDA/ARS.

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Figure 1. TEM images of antibody-functionalized silica-encapsulated nanotags: (A)

270

monomer and (B) trimer.

271

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Figure 2. SERS immunoassays were performed in 20% fetal bovine serum and contained

274

all three reporter nanotags and all three capture magnetic nanoparticles specific for WNV

275

E, RVFV N and F1 antigens. (A) All three antigens: WNV E, RVFV N and F1 (B) WNV

276

E antigen only(C) RVFV N antigen only (D) F1 antigen only and (E) No antigen present

277

(negative control). All immunoassays contained 10 ng/ml of antigen except the negative

278

control. kcts/s = 1000 counts/s.

279 280

Figure 3. SERS spectra for triplex immunoassays of a serial dilution of the F1, RVFV N

281

and WNV E antigens. Stacked spectra correspond to concentrations of (A) 100 ng/ml, (B)

282

10 ng/ml, (C) 1 ng/ml, (D) 0.1 ng/ml, (E) 50 pg/ml, and (F) 10 pg/ml of each antigen.

283

Spectrum G is the negative control without any antigen. Compared to the negative

284

control, the characteristic peaks for a positive detection are still discernable even at

285

concentrations of 10 pg/mL (inset). kcts/s = 1000 counts/s.

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Figure 4. Dose-responsive curves for triplex antigen capture in 20% fetal bovine serum.

289

Plots of the log10 concentration of antigen versus signal intensity (area under curve =

290

AUC) of the characteristic peak(s) between (A) 400-540 cm-1 for F1 antigen, (B) 570-695

291

cm-1 for RVFV N antigen, and (C) 1065-1160 cm-1 for WNV E antigen.

292 293

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TOC Graphic

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Schematic of antigen detection in the immunoassay using magnetic nanoparticles and silica-encapsulated SERS nanotags.

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Table of Contents Categories 1. Analytical Methods 2. Food Safety and Toxicology All Figures in color format.


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Detection of multiple pathogens in serum using silica-encapsulated

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