Silver enhanced step

In addition to the solution-phase protein detection assays such as biobarcode assays, a surface-based protein detection method was developed by Niemeyer and Ceyhan (70). In this work, a biotin-labeled antibody was conjugated to DNA using streptavidin as a tinker molecule. This conjugate was hybridized to complementary DNA-AuNPs and antibody-functionalized AuNPs. These particles are allowed to form a sandwich structure with a target protein and a second antibody immobilized on a flat surface. This was followed by a silver enhancement step catalyzed by the AuNPs which resulted in a LOD of 50 fmol (200 pM). This method is conceptually similar to the corresponding surface-based scanometric detection of DNA. Further work demonstrated a similar protein detection scheme without the silver enhancement (71). Instead, multiple layers of secondary DNA-AuNPs were used to increase signal enhancement [LOD = 0.1 finol (2 pM)]. 

Fig. 5.13 Scheme of extrinsic immunoassays using Ag enhanced step, a, b Two NP probes, c SERS detection of a probes after immobilization on a chip and silver enhanced step with hydroquinone (reproduced with permission from Cao et al. 2003. Copyright 2003 American Chemical Society)... 

This step relies on the silver enhancement of colloidal gold for detection. To keep background to a minimum, sections are washed in water containing a minimum of ions—which can act as nucleation sites for the silver—before and after the enhancement. 

The ions from the buffer must be removed, or they will serve as nucleation sites for the silver and increase the background. If rinsing the samples in distilled water will damage the tissue or if the acid pH of the enhancing solution will remove antibodies with low affinities, the samples may be briefly fixed and quenched before rinsing in water and proceeding to the next steps. 

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