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

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@mail.ucf.edu, Tel: 407-882-2845, Fax: 407-882-2819.

NanoScience Technology Center

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).

Figure 1. dynamic light scattering intensities and linear regression curves of gold nanospheres (GNP) and gold nanorods.

Figure 5. The calculated numerical ratio of nanoprobe aggregates over individual nanoprobes as determined by DLS measurements: (a) 1:2.5 mixture of GNP-dAb:GNR-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.

Figure 4. DLS analysis data of individual nanoprobes and nanoprobe oligomers formed with the addition of free-PSA antigens at different concentrations.

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).

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.

What’s 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-nanorod 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.

40 nm GNP

40 nm by 10 nm GNR

Why gold nanoparticles?Key Features: Strong Light Scattering Properties 4~5 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

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 (>1 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.

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:

1~2 uL- save precious blood fluidsAcknowledgement: National Science Foundation CAREER award DMR 0552294 and NIRT award 0506531.

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

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

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

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

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

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

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

3DI 3DI

3DI

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b

c

c d

50 nm

60 nm

ba