Enhancement of immunoassay's fluorescence and detection sensitivity using three-dimensional plasmonic...

Protein detection is universal and vital in biological study and medical diagnosis (e.g., cancer detection). Fluorescent immunoassay is one of the most widely used and most sensitive methods in protein detection (Giljohann, D. A.; Mirkin, C. A. Nature2009, 462, 461-464; Yager, P.; et al. Nature2006, 442, 412-418). Improvements of such assays have many significant implications. Here, we report the use of a new plasmonic structure and a molecular spacer to enhance the average fluorescence of an immunoassay of Protein A and human immunoglobulin G (IgG) by over 7400-fold and the immunoassay's detection sensitivity by 3,000,000-fold (the limit of detection is reduced from 0.9 × 10(-9) to 0.3 × 10(-15) molar (i.e., from 0.9 nM to 300 aM), compared to identical assays performed on glass plates). Furthermore, the average fluorescence enhancement has a dynamic range of 8 orders of magnitude and is uniform over the entire large sample area with a spatial variation ±9%. Additionally, we observed that, when a single molecule fluorophore is placed at a "hot spot" of the plasmonic structure, its fluorescence is enhanced by 4 × 10(6)-fold, thus indicating the potential to further significantly increase the average fluorescence enhancement and the detection sensitivity. Together with good spatial uniformity, wide dynamic range, and ease to manufacture, the giant enhancement in immunoassay's fluorescence and detection sensitivity (orders of magnitude higher than previously reported) should open up broad applications in biology study, medical diagnosis, and others.