IOP PUBLISHING NANOTECHNOLOGY Nanotechnology 19 (2008) 435703 (10pp) doi:10.1088/0957-4484/19/43/435703 Enhanced solid-phase immunoassay using gold nanoshells: effect of nanoparticle optical properties Boris Khlebtsov 1 and Nikolai Khlebtsov 1,2,3 1 Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 13 Prospekt Entuziastov, Saratov 410049, Russia 2 Saratov State University, 83 Ulitsa Astrakhanskaya, Saratov 410026, Russia E-mail: khlebtsov@ibppm.sgu.ru Received 16 May 2008, in final form 17 August 2008 Published 22 September 2008 Online at stacks.iop.org/Nano/19/435703 Abstract Plasmon-resonant nanoparticle-labeled immunoassays provide a simple, low-cost and effective way of detecting target molecules in solutions. The optical mechanisms behind their efficiency, however, have not been addressed until now. We present the first theoretical description of nanoparticle-labeled dot immunoassay and its experimental verification with functionalized 15 nm colloidal gold nanospheres and silica/gold nanoshells (GNs). Three types of GNs, with silica core diameters of 100, 140 and 180 nm and a gold shell thickness of about 15 nm, were studied in our experiments. The fabricated markers were characterized by electron and atomic-force microscopy, UV–vis spectroscopy and dynamic light scattering. A normal rabbit serum (the target IgG molecules) and sheep antirabbit antibodies (the probing molecules) were used as a biospecific model. The minimal detection limit for IgG target molecules was about 15 ng in the case of a standard dot-assay protocol based on 15 nm colloidal gold particles conjugated with probing molecules. In contrast to this observation, a simple replacement of 15 nm gold labels by GN conjugates resulted in a drastic increase in detection sensitivity of up to 0.25 ng in the case of 180/15 nm GNs and of up to 0.5–1 ng for 100/15 and 140/15 GNs. By using the theory developed, we explained the dependences of the low detection limit, the maximal-color intensity and the probe-load saturation limit on the particle parameters. S Supplementary data are available from stacks.iop.org/Nano/19/435703 (Some figures in this article are in colour only in the electronic version) 1. Introduction A very widespread method for the visualization of biospecific interactions is the solid-phase assay [1], in which the target molecules or antigens are adsorbed on a solid carrier and then revealed with various labels conjugated to recognizing probing molecules (e.g. antibodies). Among the diverse solid-phase assay techniques, a special place is occupied by the dot assay, based on the specific staining of a sample drop adsorbed on a membrane [2] or on another substrate. The chief advantage of this method is the possibility of conducting tests without using expensive equipment or means of signal processing. 3 Author to whom any correspondence should be addressed. Contrary to, for example, solid-phase assays using scanning atomic-force microscopy [3], laser-induced scattering around a nanoabsorber (LISNA) [4] 4 or single-particle resonant-light- scattering spectroscopy [6, 7], such tests may be run under home or field conditions. Examples include known tests for early pregnancy and for narcotic substances and toxins in human blood. In the dot assay, minimal volumes of target-molecule solutions are applied to a substrate as a series of spots, thus making it possible to perform many more analyses with 4 Actually, the term LISNA [4] means a deviation of a probing laser beam because of refraction caused by heating a metal particle or cluster by a power modulated or pulsed laser beam. In a sense, this principle is close to the well- known ‘thermal lens method’ see, e.g., [5]. 0957-4484/08/435703+10$30.00 © 2008 IOP Publishing Ltd Printed in the UK 1