This document describes a research project utilizing gold nanoparticles, dynamic light scattering (DLS), and surface-enhanced Raman spectroscopy (SERS) for influenza virus detection. The project involves three parts: 1) Stabilizing monoclonal antibody conjugation to gold nanoparticles by optimizing pH and antibody concentration. 2) Screening antibodies for specificity and affinity to influenza viruses using a DLS assay. 3) Developing a homogeneous SERS-based assay for multiplexed influenza virus detection. The goal is to create a fast, accurate, quantitative, multiplexed, and point-of-care detection method for influenza viruses.

1. Influenza Virus Detection
Utilizing Gold Nanoparticles,
Dynamic Light Scattering, and
SERS
Yen Lai
Research Seminar
March 6th 2015
http://www.cdc.gov/flu/images.htm accessed on 2/21/15

2. Overview
• Introduction
• Terminology and Technique Foundation
• Research Foci
• Methods, Results, and Discussion
• Project 1 – Stabilization of Gold Nanoparticle Conjugates
• Project 2 – Antibody Screening
• Project 3 – SERS Detection
• Conclusion
• Future Work 2

3. Influenza Virus – Seasonal Flu
http://www.cdc.gov/h1n1flu/yearinreview/yir5.htm accessed on 2/21/15
3
Influenza A H3N2,
H1N1, and
influenza B

4. Influenza Virus – Pandemic Flu
4
http://gamapserver.who.int/h1n1/qualitative_indicators/atlas.html?indicator=i0&date=Week
%2029%20(19-Jul-2010%20:%2025-Jul-2010) accessed on 2/15/15
2009 H1N1 Influenza Pandemic (Apr 2009 – Aug 2010)
• 284,000 deaths including 201,200 respiratory deaths,
cardiovascular disease 83,300 deaths associated with H1N1 infections
• 80% younger than 65
Nov 2009

5. Ultimate goal
Fast, accurate, quantitative, multiplexed,
and point-of-care (POC) detection
5

6. Immunoassays
• Using antibody (or immunoglobulin) to detect antigen
• Selectively target: DNA, protein, antibody, pathogen (e.g.
virus), and hormone
• First Appearance in 1950’s (Yalow and Berson)
• High specificity (antibody-antigen recognition), high-
throughput, and high sensitivity for a wide range of
analytes in biological samples
• E.g. pregnancy dipstick
6
http://www.cytodiagnostics.com/store/pc/Lateral-Flow-
Immunoassays-d6.htm access on 2/21/15
Gotcha
virus

7. Antibody-Antigen Interaction
7
Monoclonal
Antigen
Polyclonal
Hemagglutinin (HA)
Neuraminidase (NA)
Antibody
http://www.cdc.gov/flu/images.htm accessed on 2/21/15

8. Lateral Flow Assays
• Merits
• Simple (dip and read)
• Cheap ($7 - 19)
• Fast (matter of minutes)
• Not ideal for infectious
disease detection
• Not multiplexed
• Poor detection limit
8
http://www.cytodiagnostics.com/store/pc/Lateral-Flow-Immunoassays-d6.htm accessed on
2/21/15

9. Immunoassay Formats
• Heterogeneous
• Homogeneous
9
mix
Incubation Incubation
Incubation
Washing Washing

10. Labeling and Detection
10
Label type Qualities Immunoassay
formats
Sensitivity Fast Multiplexed POC Hetero. Homo.
Radioactive
labels
Yes No No No Yes No
Enzymes Yes No No No Yes No
Fluorescence
probes
Yes Yes Yes No Yes Yes
Gold
nanoparticles +
Raman reporter
Yes Yes Yes Yes Yes Yes
Labeling

11. Raman Spectroscopy
• Complement of IR Spectroscopy
• Vibrational, and rotational modes of
molecules
• Distinct structural Information
=> Multiplexing analysis
• Resistant to water
• Raman Effect
• Inherently weak signal
• Inelastic light scattering
• Stokes-high intensity; low E
• Anti-Stokes-low intensity; high E
11
Skoog, Douglas, et al. Fundamentals of analytical chemistry. Cengage
Learning, 2013.

12. Surface-Enhanced Raman Spectroscopy
• Abbreviation: SERS
• Enhancing Raman signals by
plasmonic coupling phenomenon
on metallic nanostructures
• Enhancement factor = E^4
12
Metallic Sphere
Hill, R. T., Mock, J. J., Urzhumov, Y., Sebba, D. S., Oldenburg, S. J., Chen, S. Y., ... & Smith, D. R. (2010). Leveraging nanoscale plasmonic modes to achieve reproducible
enhancement of light. Nano letters, 10(10), 4150-4154.
Au Au Au

13. Surface-Enhanced Raman Spectroscopy
• Sensitivity
• Pico/femtomolar detection limits
• Detection of a single binding event
• Multiplexing
• Narrow Raman bands, allowing use of multiple labels
• Fingerprinting analyte of interest
• Versatility
• Not sensitive to environment
• Minimal photo-bleaching
• Hardware Simplicity
• Excitation wavelength is substrate-dependent,
requiring a single excitation source
• Handheld instruments – Field Deployment!!!
13
http://www.wysri.com/wp-content/uploads/2014/08/cbex_test.png
accessed on 2/27/15

14. General Scheme
1. Gold-nanoparticle homogeneous assay
14ERL: Extrinsic Raman Label
ERL or
AuNP
probesAntibody
Raman
Reporter
virus Aggregate
Unbound ERL

15. General Scheme
2. SERS detection
15
Capillaryaction
filtering

16. 16
General Scheme
2. SERS detection
Raman
Reporter
VS.
Extrinsic
Intrinsic
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
400 900 1400
Intensity
Raman Shift (cm-1)

17. General Scheme
17
3. Multiplexed detection
Dougan, Jennifer A., and Karen Faulds. Analyst 137.3 (2012): 545-554.

18. Research Foci
• Project 1 - Stabilization of monoclonal antibody (mAb)
conjugation on AuNP
• pH – dependent adsorption
• Concentration – dependent adsorption
• Project 2 - Antibody screening
• Specificity and affinity of antibody-virus binding
• Bioactivity changes after adsorption
• Project 3 - Homogeneous assay-SERS detection 18

19. Project 1: Stabilization of Ab-AuNP
Conjugation
• Direct adsorption of antibody (Ab) on
AuNP
• NaCl is needed to keep Ab’s 3-D
structure and bioactivity
• AuNP (negatively charged) aggregates
in salt
• Full coverage of Ab can protect AuNP
from aggregation in salt
• Parameters:
• Concentration
• pH
Au
19
Na+>>>
Au

20. DLS to Monitor AuNP Conjugation
20
Dynamic Light Scattering
(DLS) readout
Color change
Au
Antibody
a
a+ 20 nm
AuNP
probes

21. DLS to Monitor AuNP Conjugation
• DLS: dynamic light scattering
• Hydrodynamic radius ⇔ diffusion velocity ⇔ fluctuation of
scattering light
• Brownian Motion
21
D is the diffusion velocity of the particle,
k is the Boltzmann constant,
T is the temperature,
η is the viscosity of the solution,
a is the hydrodynamic radius of the particle.

22. pH-Dependent Adsorption
• Citrate-caped AuNP, negatively charged
• Antibodies: charged amino acids (-COO-
and -NH3
+ ) and H-bonds
• pH influences net charge and
conformation (tertiary and quaternary
structure) of proteins/antibodies
22
Au
PDB:
3I40+

23. Experimental - pH Study of Ab Conjugation
• Antibodies: Mouse monoclonal anti-influenza A antibodies (InA88, InA97, InA4, and InA16)
specific to native HA from influenza virus A/New Caledonia/20/99 (H1N1) were purchased from
Novus Biological. The antibodies were purified by protein A affinity chromatography and supplied in
PBS, pH 7.4.
100 µL
60nm
AuNP
pH
range:
5.5, 6.5,
7.5,
8.5, 9.5
30
µg/mL
antibody
DLS
10 µL
10%
NaCl
DLS
23

24. Results – pH study of Ab Conjugation
pH 5.5, 6.5, 7.5, 8.0, 8.5, 9.5, the concentration is fixed at 30 µg/ml, monoclonal
antibodies InA97, InA88, InA4 and InA16
24
pH
5 6 7 8 9 10
MeanHydrodynamicDiameter(nm)
0
50
100
150
200
250
300
350
400
Before Salt
After Salt
pH
5 6 7 8 9 10
MeanHydrodynamicDiameter(nm)
0
50
100
150
200
250
300
350
400
InA97
InA16
InA88
InA4
InA4

25. Concentration-Dependent Adsorption
• Optimal antibody concentration: the
smallest amount of antibody to fully
coat and protect AuNP from
aggregation in salt
• Concentration ⇔ Orientation
• Certain more preferable conformation
for adsorption
• Number of contacts/antibody/unit
area
25
Au
Au
Au

26. Experimental – Concentration Study
• Antibodies: Mouse monoclonal anti-influenza A antibodies (InA4, InA16, InA88, and InA97)
specific to native HA from influenza virus A/New Caledonia/20/99 (H1N1) were purchased from
Novus Biological. The antibodies were purified by protein A affinity chromatography and supplied in
PBS, pH 7.4.
100 µL
60nm
AuNP
Optimal
pH
0 to 110
µg/mL
Antibody
DLS
10 µL
10%
NaCl
DLS
26

27. Results - Antibody Concentration Study
Antibody InA4, InA16, In88, In97,
concentration of 0 to 110 µg/ml
27
Antibody Concentration ( g/mL)
0 20 40 60 80 100
MeanHydrodynamicDiameter(nm)
0
200
400
600
800
1000
InA97
InA88
InA16
InA4

28. Protocol for mAb-AuNP Stabilization
• pH optimization
• Concentration optimization
• BSA (bovine serum
albumin) as a second
stabilizer
28
100 µL
60nm
AuNP
Optimal
pH
Optimal
antibody
concentra-
tion
33.3 µL
1% BSA
in buffer
(optimal
pH)
3 x
centrifug-
ation
10 µL
10%
NaCl
Stable Ab-AuNP conjugate in salt solution for
all the antibodies (antibody layer: ̴ 10 nm)

29. Roadmap
Project 1: stabilize Ab-AuNP
Project 2: screen antibody
Project 3: multiplexed detection
using SERS
29

30. Project 2: Antibody Screening
• WHY?
• Relation between specificity and affinity of antibody towards
antigen and assay performance
• Limited tools for mAb screening and assessment (primarily
ELISA)
• Validation of antibody’s bioactivity after modification on NP
30

31. DLS Assay for Antibody Screening
• Preliminary studies:
• S. S. Dasary et. al. ACS applied materials & interfaces, 2010, 2, 3455-3460
• H. Jans et. al. Analytical chemistry, 2009, 81, 9425-9432
• X. Liu et. al. Journal of the American Chemical Society, 2008, 130, 2780-2782
• X. Xu et. al. Analytical chemistry, 2007, 79, 6650-6654
• J. D. Driskell et al. Analyst, 2011, 136, 3083-3090
• Propose: DLS assay to investigate the specificity (no cross reaction) and
affinity (level of binding) of antibody-antigen binding
Gotcha
PR8
PR8
Mehhh
N. C.
31or

32. DLS vs. ELISA
DLS assay ELISA
Single step Multiple step
Reproducible Irreproducible
Fast (30 min) Time-consuming (24 hr)
AuNP substrate Polystyrene microtiter plate
substrate
32

33. DLS for Antibody Screening
33
sizing readout
by DLS
High affinity
Low affinity

34. Experimental
Immunoassay Protocol
34
control
PBS
1/4 1/42 1/43 1/44 1/45 1/46
Step 1: Virus
Serial Dilutions
Step 2: Adding
AuNP probes 1/4 1/42 1/43 1/44 1/45 1/46
AuNP
probes
1/4n = dilution factor

35. Project 2 - Results and Discussion
35
Driskell, J.D., et al., One-step assay for detecting influenza virus using dynamic light scattering and gold nanoparticles. Analyst, 2011. 136(15): p. 3083-3090.
InA97 vs. Human influenza A/New
Caledonia/20/99 (H1N1)
Virus Concentration (pfu/mL)
1e+2 1e+3 1e+4 1e+5 1e+6 1e+7
MeanHydrodynamicDiameterIncrease(nm)
0
20
40
60
80
100
UGA New Cal vs InA97
UIUC New Cal vs InA97
Hook point
B
C
D
Lai, Y. H., et. al., Rapid Screening of Antibody-Antigen Binding using Dynamic Light Scattering (DLS) and Gold Nanoparticles. Analytical Method, under review.
Level of aggregation = D aggregate – D free AuNP

36. Project 2 - Results and Discussion
36
Virus strain: Human influenza A/New Caledonia/20/99 (H1N1)
Mouse anti-influenza monoclonal antibody (InA4, InA16, InA88, and InA97)
Lai, Y. H., et. al., Rapid Screening of Antibody-Antigen Binding using Dynamic Light Scattering (DLS) and Gold Nanoparticles. Analytical Method, under review.
Virus Concentration (TCID50/mL)
1e+3 1e+4 1e+5 1e+6 1e+7
MeanHydrodynamicDiameter
Increase(nm)
-20
0
20
40
60
80
100
InA4
InA16
InA88
InA97
DLS – 30 min
Antibody Dilution Factor
1e+1 1e+2 1e+3 1e+4 1e+5 1e+6 1e+7
O.D.(450nm)
0.0
0.1
0.2
0.3
0.4
0.5
InA97
InA4
InA16
InA88
ELISA – 24 hr

37. Project 2 - Results and Discussion
37
Lai, Y. H., et. al., Rapid Screening of Antibody-Antigen Binding using Dynamic Light Scattering (DLS) and Gold Nanoparticles. Analytical Method, under review.
Virus Concentration (TCID50/mL)
1e+3 1e+4 1e+5 1e+6 1e+7
MeanHydrodynamicDiameter
Increase(nm)
-20
0
20
40
60
80
100
InA4
InA16
InA88
InA97
A/New Caledonia/20/99 (H1N1) A/Puerto Rico/8/34 (H1N1)
Virus Concentration (TCID50/mL)
1e+3 1e+4 1e+5 1e+6 1e+7
MeanHydrodynamicDiameter
Increase(nm)
0
20
40
60
80
100
A16
A4
A97
Verified by ELISA

38. Conclusion/Roadmap
Project 1:
- Gain understanding about the behaviors of different monoclonal antibody in the conjugation
process on AuNP
- Obtain a straightforward protocol for Ab-AuNP conjugation
Project 2:
- Establish a simple and rapid method for Ab screening using AuNP and DLS
- Select one mAb (InA97) highly specific to New Caledonia (H1N1) strain
Project 3: Multiplexed detection using SERS (current + future work) 38

39. Detection via DLS vs. SERS
39
Before filtering
DLS
After filtering
SERS
Aggregates in
solution
A

40. Detection via DLS vs. SERS
• Preliminary data (Arielle’s work):
Mouse IgG and goat anti mouse-IgG
40
DLS SERS

41. Future Work- Multiplexed detection using
SERS
41
virus
Capillaryaction
filtering
Frequency
Signal

42. References
• Schnitzler, S. U., & Schnitzler, P., Virus genes,2009, 39, 279-292.
• H. R. Hoogenboom, Nature Biotechnology, 2005, 23, 1105-1116.
• F. Ylera, S. Harth, D. Waldherr, C. Frisch and A. Knappik, Analytical Biochemistry, 2013, 441,
208-213.
• S. S. Hall and P. S. Daugherty, Protein Science, 2009, 18, 1926-1934.
• M. O'sullivan, J. Bridges and V. Marks, Annals of Clinical Biochemistry: An International
Journal of Biochemistry in Medicine, 1979, 16, 221-239.
• A. Voller, D. Bidwell and A. Bartlett, Bulletin of the World Health Organization, 1976, 53, 55.
• S. S. Dasary, D. Senapati, A. K. Singh, Y. Anjaneyulu, H. Yu and P. C. Ray, ACS Applied
Materials & Interfaces, 2010, 2, 3455-3460.
• H. Jans, X. Liu, L. Austin, G. Maes and Q. Huo, Analytical chemistry, 2009, 81, 9425-9432.
• X. Liu, Q. Dai, L. Austin, J. Coutts, G. Knowles, J. Zou, H. Chen and Q. Huo, Journal of the
American Chemical Society, 2008, 130, 2780-2782.
• G. T. Hermanson, Bioconjugate techniques, Academic press, 2013.
• J. D. Driskell, C. A. Jones, S. M. Tompkins and R. A. Tripp, Analyst, 2011, 136, 3083-3090.
42