nanoDLSA: A Novel Homogeneous Immunoassay for Biomarker Detection using Gold Nanoparticles Coupled with Dynamic Light Scattering Detection

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nanoDLSA A Novel Homogeneous Immunoassay for
Biomarker Detection using Gold Nanoparticles
Coupled with Dynamic Light Scattering Detection
NanoScience Technology Center
Xiong Liu, Qiu Dai, Lauren Austin, Janelle
Coutts, Genevieve Knowles, Jianhua Zou, Hui Chen,
Qun Huo Nanoscience Technology Center,
Department of Chemistry, University of Central
Florida, 12424 Research Parkway Suite 400,
Orlando, FL 32826 Email qhuo_at_mail.ucf.edu, Tel
407-882-2845, Fax [phone].
Whats new for cancer biomarker early detection
and diagnosis? A one-step homogeneous
immunoassay for the detection of a prostate
cancer biomarker, free-PSA (Prostate Specific
Antigen), was developed using gold nanoparticle
probes coupled with dynamic light scattering
(DLS) measurements due to their orders of
magnitude stronger light scattering properties. A
spherical gold nanoparticle (GNP) and a gold
nanorod (GNR) were first conjugated with two
different primary antibodies and then used as
optical probes for the immunoassay. In the
presence of antigen f-PSA in solution, the
nanoparticles and nanorods aggregate together
into pairs and oligomers through the formation of
a sandwich type antibody-antigen-antibody
linkage. The relative ratio of nanoparticle-nanoro
d pairs and oligomers versus individual
nanoparticles was quantitatively monitored by DLS
measurement. A correlation can be established
between this relative ratio and the amount of
antigen in solution. f-PSA in the concentration
range from 0.1 to 10 ng/mL was detected by this
one-step and washing-free homogeneous
immunoassay.

• Why gold nanoparticles?
• Key Features
• Strong Light Scattering Properties
• 45 orders higher than proteins in solutions
• High absorption efficacy at SPR band
• Biocompatible
• Photothermal conversion properties
• Ultra-sensitive detection in DLS
• GNP 0.02 pM
• GNR 0.4 pM

40 nm GNP

• How it works?
• Mix of biomarker solution with nanoprobes
  solution and incubate
• Measurement of Sizes and Size Distributions of
  nanoprobes and their oligomers by DLS
• Analysis and processing of data
• Numerical ratio of oligomers over individual
  nanoprobes obtained
• Different biomarker concentration shows
  different ratio
• Level of biomarker message presented

50 nm
40 nm by 10 nm GNR
60 nm
Figure 1. dynamic light scattering intensities
and linear regression curves of gold nanospheres
(GNP) and gold nanorods.
Figure 4. DLS analysis data of individual
nanoprobes and nanoprobe oligomers formed with
the addition of free-PSA antigens at different
concentrations.

• Dynamic Light Scattering Detector
• Trace Brownian motion of particles in solution
• Measure diffusion constants of particles in
  solution after Fast Fourier Transform (FFT)
• Decoding of hydrodynamic diameter information of
  particles by Stokes-Einstein Equation.
• Traditional Use Qualitative
• Protein size measurement (gt1 ug/mL)
• Trace for Protein aggregates and glycoprotien
  formation for proteins and drugs
• New Feature Quantitative, Ultra-Sensitive
• To trace cancer biomarker levels in pg/mL range
• Significant amplification of signal with
  nanotechnology-embedded probes.

• Surface Plasmon Resonance
• Gold nanospheres 532 nm
• Gold nanorods 520 nm and 730 nm
• Red-shift of bands after antibody conjugation
  indicated successful conjugation process

• The Reaction
• Biomarkers initialize sandwich of GNP and GNR
• Cross-link due to multiple antibodies on
  nanoprobes
• Reaction induce hydrodynamic diameter increase
  of nanoprobes
• More biomarker, more oligomers, higher numerical
  ratio

a

Figure 2. UV-Vis spectra of gold nanoparticles
and gold nanorods and their conjugates with
primary antibodies (a) citrate-protected gold
nanoparticles (GNP) (b) f-PSA detector antibody
conjugated gold nanoparticles (GNP-dAb) (c)
CTAB-protected gold nanorods (GNR) and (d) f-PSA
capture antibody conjugated gold nanorods
(GNR-cAb).
b

• Immunoassay
• free-prostate specific antigen
• Assay range 0-10 ng/mL
• Increasing tendency observed
• Sensitivity better than ELISA
• Specificity verified

• Direct View of
  Reaction Oligomers
• Dimmer, trimmer, tetramer and oligomers for GNP
  and GNR pairs visualized by HRTEM
• Conjugation activity was further verified by
  2oAb-conjugated 5 nm GNPs

• High Sensitivity for Aggregates
• Three order of magnitude more sensitive in
  aggregates formation than individual particles

b
a

• Key Advantages
• High sensitivity pg/mLto ng/mL range
• Washing free- versus multiple washing and
  incubation in ELISA
• Homogeneous- much better reactivity
• Fast- 15 minutes for assay is reachable
• Extremely small sample volume 12 uL- save
  precious blood fluids

• Conjugation of Antibodies to Nanoprobes
• Detector antibody- GNPs
• Capture antibody- GNRs
• Size increase from surface antibody layer was
  monitored by DLS
• GNP Nanoprobe 56.7 nm
• GNR Nanoprobe 37.2 nm
• After mixed, ready for immunoassay

c
c
d
Acknowledgement National Science Foundation
CAREER award DMR 0552294 and NIRT award 0506531.
Figure 5. The calculated numerical ratio of
nanoprobe aggregates over individual nanoprobes
as determined by DLS measurements (a) 12.5
mixture of GNP-dAbGNR-cAb in the presence of
f-PSA 1.0 ng/mL (b) measurements at different
f-PSA level (the unknown sample has a
concentration of 0.5 ng/ml, data labeled with an
asterisk) and (c) specificity and cross
reactivity test with biomarker CA125.

• References
• Liu, X. Dai Q. Austin, L. Coutts, j. Knowles,
  G. Zou, J. Chen, H. Huo, Q. A one-step
  homogenesou immunoassay for cancer biomarker
  detection using gold nanoparticle probes coupled
  with dynamic light scattering. J. Am. Chem. Soc.
  2008,130, 2780-2782.
• Liu, X. Atwater, M. Wang, J. Huo, Q.
  Extinction coefficient of gold nanoparticles with
  different sizes and different capping ligands.
  Colloids and Surface B Biointerfaces 2007, 58,
  3-7.

Figure 3. TEM micrographs of (a-c) nanoparticle
oligomers formed from a mixture of primary
antibodies conjugated gold nanoparticles and gold
nanorods with the addition of f-PSA antigens (2
ng/mL) in the mixed nanoprobe solution and (d-f)
same nanoparticle oligomers, but with additional
conjugations of 2nd antibody-coated 5 nm gold
nanoparticles to the oligomers (Scale bar 20 nm,
except for d, which is 10 nm).