实验概要

The surface-confined assay format is one of the most convenient detection formats used in many immunoassays. Fluorescence emission from monolayers of dyes requires a strong excitation and good detection system. Such samples are susceptible to artifacts due to background fluorescence from substrates. We demonstrate that using silver nanostructures deposited on the slide substrate can significantly enhance measured fluorescence, reduce unwanted background and increase photostability of the used probes. Using thin layers of polymer doped with fluorescein, we tested two nanostructures—silver island films (SIFs) deposited on glass slides and self-assembled colloidal structures (SACS) deposited on thin silver film. The SACS surfaces show extraordinary fluorescence enhancements: over 100-folds in hot spots. We applied these surfaces for enhanced Alexa488 model immunoassay.

实验原理

1. Metal-Enhanced Fluorescence

Discovery of surface-enhanced Raman scattering (SERS) (1, 2) revitalized Raman spectroscopy and resulted in many new applications of the technology. In contrast to SERS, observed enhancement of fluorescence signal is not significant and often controversial due to strong quenching in a close proximity to the metal surface.

Metal-enhanced fluorescence (MEF) was demonstrated already three decades ago (3–6). Recently, several groups reported fluorescence enhancements on various silvered surfaces, including silver island films (SIFs) (7, 8), deposited colloids (9, 10), photodeposited structures (11), and electron beam-deposited nanostructures (12). Although the enhancements were modest, usually in the order of tenfold as compared to many thousandfolds in SERS, the dependence of the enhancement on the distance from the metallic surface to fluorophores has been established (9, 13). In general, the strongest enhancements are observed between 40 and 200 Å away from the metallic surface. At shorter distance the quenching dominates the fluorophore–metal interactions and at longer distances the enhancement gradually decreases. There are two enhancement effects: first is the enhanced local field, which is responsible for a higher excitation rate; and a second enhancement effect is due to an interaction of an excited molecule dipole with the nanoparticles, known as radiative decay engineering (RDE), which is responsible for a decrease of fluorescence lifetime. Total enhancement is the product of these two effects. The recent reviews (14, 15) contain more details on MEF.

2. Model Immunoassays

To demonstrate a metal-enhanced fluorescence immunoassay on the glass surface coated with SIF, we used a model immunoassay format shown in Scheme 1. A model antigen, mouse Immunoglobulin G (IgG), was immobilized on the surfaces (SIF-coated surface and glass surface used as a reference), and corresponding specific antibodies labeled with a fluorophore were allowed to bind to the antigen. We incubated samples with antibodies for about 1 h to complete the binding reaction. Then the non-bound labeled antibodies were removed, a buffer was added, and fluorescence signal was measured in front-face configuration.

The measured fluorescence signal was a result of a specific interaction. To verify this, we performed the immunoassay using the same labeled antibodies, but with a nonspecific antigen, rabbit IgG, immobilized on the surface instead of the specific antigen. When a nonspecific antigen was used for the model immunoassay, no significant increase of the signal (non-specific binding) was observed. For more information about fluorescent immunoassays we refer reader to the recent review (16).

We found that silver colloids deposited on metallic mirrors show stronger enhancements than those deposited on glass substrates (17,18). It has been established that enhancements on sharp metallic edges are significantly more efficient (19–21). Recently, we reported exceptionally strong local enhancements on self-assembled colloidal structures (SACS) formed on metallic films (22). This effect allows a significant reduction of the excitation intensity and effectively eliminates unwanted background from the regions away from the surface. The SACS looks similar to fractal-like structures with sharp edges. The interaction of local plasmons with metallic film results in strong local fields. A similar effect was recently described for a nanowire deposited on a metal film as an efficient platform for SERS (23).

In this chapter we will compare the fluorescence enhancements on SIFs and SACS. We will also emphasize the advantages of SACS in a model Alexa488 immunoassay. The immunoassays are ideal systems for observing strong fluorescence enhancements because the fluorescent probe is located at a close distance from the metallic surface.

主要试剂

1. PVA Films Doped with Fluorescein

Laser Grade Disodiumfluorescein was from Exction, Inc. Low molecular weight (13,000–23,000 MW) poly(vinyl alcohol) (PVA) was from Aldrich. Fluorescein-doped PVA samples were prepared on 1 × 1 in. microscope cover slips, either SIF coated or with SACS prepared on silver film.

2. Alexa488 Model Immunoassay

Mouse and rabbit IgGs, buffer components and salts (such as bovine serum albimun, sucrose) were from Sigma-Aldrich. Bocking solution was 1% bovine serum albumin, 1% sucrose, 0.05% NaN₃, 0.05% Tween-20 in 50 mM Na-phosphate buffer, pH 7.3. Goat-anti-mouse antibodies labeled with AlexaFluor-488 were from Invitrogen, Inc. (dye/protein ratio 7). Microscope glass slides, 3 × 1 in., and 1 mm thick, were from VWR. Water was purified by Milli-Q system.

3. SIFs and SACS

Trisodium citrate was supplied by Spectrum. Silver nitrate and glucose were from Sigma-Aldrich. Microscope slides were coated by EMF Corp. (Ithaca, NY). A 52-nm thick layer of silver was deposited on the slide with about 2-nm chromium undercoat. The silver films were protected with 5 nm layer of silica. Poly-l-lysine solution was freshly prepared solution: 8 ml water   1.0 ml Na–phosphate buffer, 50 mM, pH 7.4,  1.0 ml poly-l-lysine solution (0.1%, Sigma).