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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: [email protected], Tel: 407-882-2845, Fax: 407-882-2819. Why gold nanoparticles? Key Features: What’s new for cancer biomarker early detection and diagnosis? 40 nm GNP Strong Light Scattering Properties 4~5 orders higher than proteins in solutions High absorption efficacy at SPR 50 nm band Biocompatible Photothermal conversion properties 40 nm by 10 nm GNR Ultra-sensitive detection in DLS GNP: 0.02 pM GNR: 0.4 pM 60 nm Figure 1. dynamic light scattering intensities and linear regression curves of gold nanospheres (GNP) and gold nanorods. I D3 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 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). New Feature: Quantitative, Ultra-Sensitive High Sensitivity for Aggregates Three order of magnitude more sensitive in aggregates formation than individual particles ID To trace cancer biomarker levels in pg/mL range Significant amplification of signal with nanotechnology-embedded probes. a b 3 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 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 b Immunoassay free-prostate specific antigen Assay range: 0-10 ng/mL Increasing tendency observed Sensitivity better than ELISA Specificity verified Key Advantages Conjugation of Antibodies to Nanoprobes c d Acknowledgement: National Science Foundation CAREER award DMR 0552294 and NIRT award 0506531. 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). 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 Gold nanospheres: 532 nm Gold nanorods: 520 nm and 730 nm Red-shift of bands after antibody conjugation indicated successful conjugation process Dimmer, trimmer, tetramer and oligomers for GNP and GNR pairs visualized by HRTEM Conjugation activity was further verified by 2oAb-conjugated 5 nm GNPs 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 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 nanoparticlenanorod 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. Surface Plasmon Resonance Direct View of Reaction: Oligomers How it works? 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. 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 fluids c 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.