Detection of Multiple Pathogens in Serum Using Silica-Encapsulated

May 7, 2018 - With these unique merits enabling high-level multiplex analysis,(7−9) ... on the nanotags improves the performance of SERS-based bioas...
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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).

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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

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

15

West Nile Virus, Rift Valley Fever Virus, Yersinia pestis

16 17 18

INTRODUCTION

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

20

detection modality for both portable diagnostic tools and high throughput laboratory

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1, 2

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analysis has garnered significant attention over the past two decades

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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

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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

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

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

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

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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

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biocompatibility, stability, optical transparency and ease of surface modification

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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

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

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silica shell via Stöber’s method

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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

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

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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

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

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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

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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

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

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detection of pathogenic antigens during infection has great significance in epidemic

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

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

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

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

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. In this

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

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required hours of preparation

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

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

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

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

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

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

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under an hour with no sample preparation.

33

. In another advancement, the simultaneous detection of

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MATERIALS AND METHODS

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

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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,

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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-

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

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

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

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

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

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filtration units before use.

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

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

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

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

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

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

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

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

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

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

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

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

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chemiluminescence detection.

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

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magnetic nanoparticles were synthesized as previously reported 18 with slight

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

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

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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adjusted to a neutral pH.

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

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

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

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

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

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

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

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

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

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

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

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

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were resuspended in PBS at a desired concentration for multiplexing.

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

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

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

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

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

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

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

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

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

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

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

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

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acquisitions (1 s integration time).

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Results and discussion

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

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target antigen detection using capture magnetic nanoparticles and reporter nanotags.

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

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

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

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

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

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

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

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

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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

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

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as serum samples where heterogeneous proteins co-exist.

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

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

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

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

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

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

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

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

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

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intensity better than a single encapsulated nanotag 39.

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

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

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

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

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

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distinguishable Raman signals with similar intensity and minimal overlap in the

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

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analytes (Figure 2).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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nanotags and capture magnetic nanoparticles reagents only demonstrate negligible

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background peaks (spectrum E).

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

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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-

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

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

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

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

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

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(Spectrum F) in 20% fetal bovine serum.

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

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

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

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

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

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

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

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

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stoichiometric ratio of reporter nanotags and capture magnetic nanoparticles are currently

240

under investigation.

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

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can simultaneously detect three antigens in simulated serum samples by magnetic

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aggregation

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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

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. In

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

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

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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

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

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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

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

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

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

257

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

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that is specially engineered for magnetic capture of nanotags.

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260

Acknowledgements

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

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(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)

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monomer and (B) trimer.

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

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

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

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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)

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

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

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

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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.

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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

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

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References

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1. Driscoll, A. J.; Harpster, M. H.; Johnson, P. A., The development of surface-enhanced Raman scattering as a detection modality for portable in vitro diagnostics: progress and challenges. Physical Chemistry Chemical Physics 2013, 15 (47), 20415-20433.

297 298

2. Laing, S.; Gracie, K.; Faulds, K., Multiplex in vitro detection using SERS. Chemical Society Reviews 2016, 45 (7), 1901-1918.

299 300 301

3. Sun, L.; Yu, C. X.; Irudayaraj, J., Surface-enhanced raman scattering based nonfluorescent probe for multiplex DNA detection. Analytical Chemistry 2007, 79 (11), 3981-3988.

302 303

4. Wang, H. N.; Vo-Dinh, T., Multiplex detection of breast cancer biomarkers using plasmonic molecular sentinel nanoprobes. Nanotechnology 2009, 20 (6), 6.

304 305

5. Kang, T.; Yoo, S. M.; Yoon, I.; Lee, S. Y.; Kim, B., Patterned Multiplex Pathogen DNA Detection by Au Particle-on-Wire SERS Sensor. Nano Letters 2010, 10 (4), 1189-1193.

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Page 17 of 20

Journal of Agricultural and Food Chemistry

306 307 308

6. Shen, A. G.; Chen, L. F.; Xie, W.; Hu, J. C.; Zeng, A.; Richards, R.; Hu, J. M., Triplex AuAg-C Core Shell Nanoparticles as a Novel Raman Label. Advanced Functional Materials 2010, 20 (6), 969-975.

309 310 311

7. Graham, D.; Mallinder, B. J.; Whitcombe, D.; Watson, N. D.; Smith, W. E., Simple multiplex genotyping by surface-enhanced resonance Raman scattering. Analytical Chemistry 2002, 74 (5), 1069-+.

312 313

8. Faulds, K.; Smith, W. E.; Graham, D., Evaluation of surface-enhanced resonance Raman scattering for quantitative DNA analysis. Analytical Chemistry 2004, 76 (2), 412-417.

314 315 316

9. Porter, M. D.; Lipert, R. J.; Siperko, L. M.; Wang, G.; Narayanana, R., SERS as a bioassay platform: fundamentals, design, and applications. Chemical Society Reviews 2008, 37 (5), 1001-1011.

317 318

10. Li, Y. G.; Cu, Y. T. H.; Luo, D., Multiplexed detection of pathogen DNA with DNA-based fluorescence nanobarcodes. Nature Biotechnology 2005, 23 (7), 885-889.

319 320 321

11. Stokes, R. J.; Macaskill, A.; Lundahl, P. J.; Smith, W. E.; Faulds, K.; Graham, D., Quantitative enhanced Raman scattering of labeled DNA from gold and silver nanoparticles. Small 2007, 3 (9), 1593-1601.

322 323 324 325

12. Jun, B. H.; Noh, M. S.; Kim, J.; Kim, G.; Kang, H.; Kim, M. S.; Seo, Y. T.; Baek, J.; Kim, J. H.; Park, J.; Kim, S.; Kim, Y. K.; Hyeon, T.; Cho, M. H.; Jeong, D. H.; Lee, Y. S., Multifunctional Silver-Embedded Magnetic Nanoparticles as SERS Nanoprobes and Their Applications. Small 2010, 6 (1), 119-125.

326 327 328

13. Maiti, K. K.; Samanta, A.; Vendrell, M.; Soh, K. S.; Olivo, M.; Chang, Y. T., Multiplex cancer cell detection by SERS nanotags with cyanine and triphenylmethine Raman reporters. Chemical Communications 2011, 47 (12), 3514-3516.

329 330 331

14. Dinish, U. S.; Balasundaram, G.; Chang, Y. T.; Olivo, M., Actively Targeted In Vivo Multiplex Detection of Intrinsic Cancer Biomarkers Using Biocompatible SERS Nanotags. Scientific Reports 2014, 4, 7.

332 333 334

15. Lee, S.; Chon, H.; Lee, J.; Ko, J.; Chung, B. H.; Lim, D. W.; Choo, J., Rapid and sensitive phenotypic marker detection on breast cancer cells using surface-enhanced Raman scattering (SERS) imaging. Biosensors & Bioelectronics 2014, 51, 238-243.

335 336 337

16. Cam, D.; Keseroglu, K.; Kahraman, M.; Sahin, F.; Culha, M., Multiplex identification of bacteria in bacterial mixtures with surface-enhanced Raman scattering. Journal of Raman Spectroscopy 2010, 41 (5), 484-489.

338 339

17. Ravindranath, S. P.; Wang, Y. L.; Irudayaraj, J., SERS driven cross-platform based multiplex pathogen detection. Sensors and Actuators B-Chemical 2011, 152 (2), 183-190.

340 341 342

18. Neng, J.; Harpster, M. H.; Wilson, W. C.; Johnson, P. A., Surface-enhanced Raman scattering (SERS) detection of multiple viral antigens using magnetic capture of SERS-active nanoparticles. Biosensors & Bioelectronics 2013, 41, 316-321.

343 344

19. Sharma, B.; Frontiera, R. R.; Henry, A. I.; Ringe, E.; Van Duyne, R. P., SERS: Materials, applications, and the future. Materials Today 2012, 15 (1-2), 16-25.

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Page 18 of 20

345 346 347

20. Huang, J.; Kim, K. H.; Choi, N.; Chon, H.; Lee, S.; Choo, J., Preparation of SilicaEncapsulated Hollow Gold Nanosphere Tags Using Layer-by-Layer Method for Multiplex Surface-Enhanced Raman Scattering Detection. Langmuir 2011, 27 (16), 10228-10233.

348 349 350

21. Sha, M. Y.; Xu, H. X.; Natan, M. J.; Cromer, R., Surface-Enhanced Raman Scattering Tags for Rapid and Homogeneous Detection of Circulating Tumor Cells in the Presence of Human Whole Blood. Journal of the American Chemical Society 2008, 130 (51), 17214-+.

351 352 353

22. Wang, Y. L.; Seebald, J. L.; Szeto, D. P.; Irudayaraj, J., Biocompatibility and Biodistribution of Surface-Enhanced Raman Scattering Nanoprobes in Zebrafish Embryos: In vivo and Multiplex Imaging. Acs Nano 2010, 4 (7), 4039-4053.

354 355 356

23. Qian, X. M.; Peng, X. H.; Ansari, D. O.; Yin-Goen, Q.; Chen, G. Z.; Shin, D. M.; Yang, L.; Young, A. N.; Wang, M. D.; Nie, S. M., In vivo tumor targeting and spectroscopic detection with surface-enhanced Raman nanoparticle tags. Nature Biotechnology 2008, 26 (1), 83-90.

357 358 359

24. Doering, W. E.; Piotti, M. E.; Natan, M. J.; Freeman, R. G., SERS as a foundation for nanoscale, optically detected biological labels. Advanced Materials 2007, 19 (20), 31003108.

360 361 362 363

25. Schiestel, T.; Brunner, H.; Tovar, G. E. M., Controlled surface functionalization of silica nanospheres by covalent conjugation reactions and preparation of high density streptavidin nanoparticles. Journal of Nanoscience and Nanotechnology 2004, 4 (5), 504511.

364 365

26. Doering, W. E.; Nie, S. M., Spectroscopic tags using dye-embedded nanoparticles and surface-enhanced Raman scattering. Analytical Chemistry 2003, 75 (22), 6171-6176.

366 367 368

27. Mulvaney, S. P.; Musick, M. D.; Keating, C. D.; Natan, M. J., Glass-coated, analytetagged nanoparticles: A new tagging system based on detection with surface-enhanced Raman scattering. Langmuir 2003, 19 (11), 4784-4790.

369 370 371

28. Stober, W.; Fink, A.; Bohn, E., CONTROLLED GROWTH OF MONODISPERSE SILICA SPHERES IN MICRON SIZE RANGE. Journal of Colloid and Interface Science 1968, 26 (1), 62&.

372 373 374 375

29. Fernandez-Lopez, C.; Mateo-Mateo, C.; Alvarez-Puebla, R. A.; Perez-Juste, J.; Pastoriza-Santos, I.; Liz-Marzan, L. M., Highly Controlled Silica Coating of PEG-Capped Metal Nanoparticles and Preparation of SERS-Encoded Particles. Langmuir 2009, 25 (24), 1389413899.

376 377 378 379

30. Zavaleta, C. L.; Smith, B. R.; Walton, I.; Doering, W.; Davis, G.; Shojaei, B.; Natan, M. J.; Gambhir, S. S., Multiplexed imaging of surface enhanced Raman scattering nanotags in living mice using noninvasive Raman spectroscopy. Proceedings of the National Academy of Sciences of the United States of America 2009, 106 (32), 13511-13516.

380 381

31. Moore, D. M.; Jaykus, L., Virus–Bacteria Interactions: Implications and Potential for the Applied and Agricultural Sciences. Viruses 2018, 10.

382 383 384

32. Adjou Moumouni, P. F.; Aplogan, G. L.; Katahira, H.; Gao, Y.; Guo, H.; Efstratiou, A.; Jirapattharasate, C.; Wang, G.; Liu, M.; Ringo, A. E.; Umemiya-Shirafuji, R.; Suzuki, H.; Xuan, X., Prevalence, risk factors, and genetic diversity of veterinary important tick-borne pathogens

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Page 19 of 20

Journal of Agricultural and Food Chemistry

385 386

in cattle from Rhipicephalus microplus-invaded and non-invaded areas of Benin. Ticks and Tick-borne Diseases 2018, 9 (3), 450-464.

387 388 389 390

33. Wang, Z.; Yang, H.; Wang, M.; Petti, L.; Jiang, T.; Jia, Z.; Xie, S.; Zhou, J., SERS-based multiplex immunoassay of tumor markers using double SiO 2 @Ag immune probes and gold-film hemisphere array immune substrate. Colloids and Surfaces A: Physicochemical and Engineering Aspects 2018, 546, 48-58.

391 392

34. Zhang, D.; Huang, L.; Liu, B.; Ni, H.; Sun, L.; Su, E.; Chen, H.; Gu, Z.; Zhao, X., Quantitative and ultrasensitive detection of multiplex cardiac biomarkers in

393 394

lateral fl ow assay with core-shell SERS nanotags. Biosensors and Bioelectronics, 2018; Vol. 106, pp 204-211.

395 396 397

35. Driscoll, A. J.; Johnson, P. A., Numerical modeling of analyte diffusion and adsorption behavior in microparticle and nanoparticle based biosensors. Chemical Engineering Science 2018, 184, 141-148.

398 399 400 401

36. Neng, J.; Harpster, M. H.; Zhang, H.; Mecham, J. O.; Wilson, W. C.; Johnson, P. A., A versatile SERS-based immunoassay for immunoglobulin detection using antigen-coated gold nanoparticles and malachite green-conjugated protein A/G. Biosensors & Bioelectronics 2010, 26 (3), 1009-1015.

402 403 404 405

37. Drolet, B. S.; Weingartl, H. M.; Jiang, J. Y.; Neufeld, J.; Marszal, P.; Lindsay, R.; Miller, M. M.; Czub, M.; Wilson, W. C., Development and evaluation of one-step rRT-PCR and immunohistochemical methods for detection of Rift Valley fever virus in biosafety level 2 diagnostic laboratories. Journal of Virological Methods 2012, 179 (2), 373-382.

406 407 408

38. Cao, H. N.; He, J.; Deng, L.; Gao, X. Q., Fabrication of cyclodextrin-functionalized superparamagnetic Fe3O4/amino-silane core-shell nanoparticles via layer-by-layer method. Applied Surface Science 2009, 255 (18), 7974-7980.

409 410 411

39. Salehi, M.; Schneider, L.; Strobel, P.; Marx, A.; Packeisen, J.; Schlucker, S., Two-color SERS microscopy for protein co-localization in prostate tissue with primary antibodyprotein A/G-gold nanocluster conjugates. Nanoscale 2014, 6 (4), 2361-2367.

412 413 414 415

40. Wang, G. F.; Lipert, R. J.; Jain, M.; Kaur, S.; Chakraboty, S.; Torres, M. P.; Batra, S. K.; Brand, R. E.; Porter, M. D., Detection of the Potential Pancreatic Cancer Marker MUC4 in Serum Using Surface-Enhanced Raman Scattering. Analytical Chemistry 2011, 83 (7), 25542561.

416 417 418

41. Chon, H.; Lim, C.; Ha, S. M.; Ahn, Y.; Lee, E. K.; Chang, S. I.; Seong, G. H.; Choo, J., OnChip Immunoassay Using Surface-Enhanced Raman Scattering of Hollow Gold Nanospheres. Analytical Chemistry 2010, 82 (12), 5290-5295.

419 420 421 422

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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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