A Label-Free Immunoassay Based Upon Localized Surface Plasmon Resonance of Gold Nanorods Kathryn M. Mayer, † Seunghyun Lee, ‡ Hongwei Liao, ‡ Betty C. Rostro, † Amaris Fuentes, † Peter T. Scully, † Colleen L. Nehl, † and Jason H. Hafner †,‡, * † Department of Physics & Astronomy and ‡ Department of Chemistry, Rice University, Houston, Texas 77005 G old and silver nanoparticles ex- hibit localized surface plasmon resonances (LSPR) at visible and near-infrared frequencies, leading to sharp peaks in their spectral extinction. 1 The de- pendence of the resonance condition on the local dielectric environment enables a simple form of molecular sensing in which analyte binding to the nanoparticles surface causes a shift in the spectral extinction peak. 2 LSPR sensing is therefore the nano- particle analogue of surface plasmon reso- nance sensing (SPR), which similarly moni- tors the resonance condition for surface plasmons in thin gold films. 3 SPR is a pow- erful surface analytical technique since it can detect submonolayer quantities of ana- lyte at the gold film surface. Furthermore, since SPR measures an inherent property of the analyte, it does not require further label- ing or chemical amplification. SPR can therefore measure dynamic processes in real time such as binding kinetics of biomo- lecular interactions, rather than simply pro- viding the end-points. These properties have led to widespread use of SPR in the study of biomolecular interactions, as well as antibody screening for diagnostic and therapeutic applications. 4,5 However, de- spite its analytical capabilities, SPR is not widely used in clinical immunoassays or other nonresearch applications owing to the complexity of the optical instrumenta- tion and the need for precise temperature control. It has been suggested that LSPR sensing with nanoparticle substrates will preserve the virtues of SPR but greatly broaden the scientific and technological ap- plications, since LSPR sensing is based on a simple optical extinction measurement, is not temperature sensitive, and requires only common laboratory equipment. 6 Fur- thermore, nanoparticles have a highly local- ized LSPR sensing volume which eliminates the need to trap the interacting molecules of interest in a polymer matrix to enhance the signal, as is often done in SPR measurements. LSPR sensing has evolved through the research of several groups over the past de- cade. The principle was first demonstrated in 1998 with antibody-conjugated gold nanoparticles in solution. 7 To mitigate spu- rious spectral shifts due to aggregation, nanoparticles were conjugated to mono- clonal antibodies specific for a single epitope on the target ligand. However, to completely remove the possibility of aggre- gation, others have worked with nanoparti- cles bound to a transparent substrate, as demonstrated with silver nanotriangles cre- ated by nanosphere lithography, 8 and gold colloid films on glass. 9 Since these initial studies, there have been many reports on the technique 10–29 including demonstra- tions of multiplexing, 20,30 the detection of medically relevant analytes in clinical samples, 31 and fiber-based sensors. 10,13 Despite these successes, LSPR sensing is still not nearly as prevalent as SPR. Thus far, See the accompanying Perspective by Odom and Nehl on p 612. *Address correspondence to hafner@rice.edu Received for review November 18, 2007 and accepted February 11, 2008. Published online February 22, 2008. 10.1021/nn7003734 CCC: $40.75 © 2008 American Chemical Society ABSTRACT Robust gold nanorod substrates were fabricated for refractive index sensing based on localized surface plasmon resonance (LSPR). The substrate sensitivity was 170 nm/RIU with a figure of merit of 1.3. To monitor biomolecular interactions, the nanorod surfaces were covered with a self-assembled monolayer and conjugated to antibodies by carbodiimide cross-linking. Interactions with a specific secondary antibody were monitored through shifts in the LSPR spectral extinction peak. The resulting binding rates and equilibrium constant were in good agreement with literature values for an antibody–antigen system. The nanorod LSPR sensors were also shown to be sensitive and specific. These results demonstrate that given a sufficiently stable nanoparticle substrate with a well defined chemical interface, LSPR sensing yields similar results to the surface plasmon resonance technique, yet with much simpler instrumentation. KEYWORDS: gold nanorod · biosensor · immunoassay · localized surface plasmon resonance · SPR · nanobiotechnology · nanophotonics ARTICLE www.acsnano.org VOL. 2 ▪ NO. 4 ▪ 687–692 ▪ 2008 687