Influenza Virus Detection Utilizing Gold Nanoparticles, Dynamic Light Scattering, and SERS

Yen LaiResearch SeminarMarch 6th 2015

http://www.cdc.gov/flu/images.htm accessed on 2/21/15

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 Work2

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

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

Ultimate goal

Fast, accurate, quantitative, multiplexed,

and point-of-care (POC) detection

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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 dipstick6

http://www.cytodiagnostics.com/store/pc/Lateral-Flow-Immunoassays-d6.htm access on 2/21/15

Gotcha

virus

Antibody-Antigen Interaction

7

Monoclonal

Antigen

Polyclonal

Hemagglutinin (HA) Neuraminidase (NA)

Antibody

http://www.cdc.gov/flu/images.htm accessed on 2/21/15

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

Immunoassay Formats

• Heterogeneous

• Homogeneous

9

mix

Incubation Incubation

Incubation

Washing Washing

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

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. CengageLearning, 2013.

Surface-Enhanced Raman Spectroscopy• Abbreviation: SERS

• Enhancing Raman signals by plasmonic coupling phenomenonon metallic nanostructures

• Enhancement factor = E^4

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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

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!!!

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http://www.wysri.com/wp-content/uploads/2014/08/cbex_test.pngaccessed on 2/27/15

General Scheme

1. Gold-nanoparticle homogeneous assay

14ERL: Extrinsic Raman Label

ERL or AuNP

probesAntibody

Raman Reporter

virus Aggregate

Unbound ERL

General Scheme

2. SERS detection

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Cap

illar

y ac

tio

n

filtering

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

Inte

nsi

ty

Raman Shift (cm-1)

General Scheme

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3. Multiplexed detection

Dougan, Jennifer A., and Karen Faulds. Analyst 137.3 (2012): 545-554.

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

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

DLS to Monitor AuNP Conjugation

20Dynamic Light Scattering (DLS) readout

Color change

Au

Antibody

a a+ 20 nm

AuNPprobes

DLS to Monitor AuNP Conjugation

• DLS: dynamic light scattering

• Hydrodynamic radius ⇔ diffusion velocity ⇔ fluctuation of scattering light

• Brownian Motion

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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.

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

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Au

PDB:3I40+

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

DLS10 µL 10% NaCl

DLS

23

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

24pH

5 6 7 8 9 10

Me

an

Hyd

rodyn

am

ic D

iam

ete

r (n

m)

0

50

100

150

200

250

300

350

400

Before Salt

After Salt

pH

5 6 7 8 9 10

Mean H

ydro

dynam

ic D

iam

ete

r (n

m)

0

50

100

150

200

250

300

350

400

InA97

InA16

InA88

InA4

InA4

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

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Au

Au

Au

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

AntibodyDLS

10 µL 10% NaCl

DLS

26

Results - Antibody Concentration Study

Antibody InA4, InA16, In88, In97, concentration of 0 to 110 µg/ml

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Antibody Concentration ( g/mL)

0 20 40 60 80 100

Mean H

ydro

dynam

ic D

iam

ete

r (n

m)

0

200

400

600

800

1000

InA97

InA88

InA16

InA4

Protocol for mAb-AuNP Stabilization

• pH optimization

• Concentration optimization

• BSA (bovine serum albumin) as a second stabilizer

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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)

Roadmap

Project 1: stabilize Ab-AuNP

Project 2: screen antibody

Project 3: multiplexed detection using SERS

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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

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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

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

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DLS for Antibody Screening

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sizing readout by DLS

High affinity

Low affinity

ExperimentalImmunoassay Protocol

34

control

PBS1/4 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

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

Mean H

ydro

dynam

ic D

iam

ete

r In

cre

ase (

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

Project 2 - Results and Discussion

36Virus 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

Me

an

Hyd

rodyn

am

ic D

iam

ete

rIn

cre

ase

(n

m)

-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

. (4

50 n

m)

0.0

0.1

0.2

0.3

0.4

0.5InA97

InA4

InA16

InA88

ELISA – 24 hr

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

Mean H

ydro

dynam

ic D

iam

ete

rIn

cre

ase (

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

Mean H

ydro

dynam

ic D

iam

ete

rIn

cre

ase (

nm

)

0

20

40

60

80

100

A16

A4

A97

Verified by ELISA

Conclusion/RoadmapProject 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

Detection via DLS vs. SERS

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Before filteringDLS

After filteringSERS

Aggregates in solution

A

Detection via DLS vs. SERS• Preliminary data (Arielle’s work):

Mouse IgG and goat anti mouse-IgG

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DLS SERS

Future Work- Multiplexed detection using SERS

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virusC

apill

ary

acti

on

filtering

Frequency

Sign

al

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.

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