Myoglobin Immunoassay Utilizing Directional Surface Plasmon-Coupled Emission

Abstract

We described an immunoassay for the cardiac marker myoglobin on a thin silver mirror surface using surface plasmon-coupled emission (SPCE). SPCE occurs for fluorophores in proximity (within approximately 200 nm) of a thin metal film (in our case, silver) and results in a highly directional radiation through a glass substrate at a well-defined angle from the normal axis. We used the effect of SPCE to develop a myoglobin immunoassay on the silver mirror surface deposited on a glass substrate. Binding of the labeled anti-myoglobin antibodies led to the enhanced fluorescence emission at a specific angle of 72 degrees . The directional and enhanced directional fluorescence emission enables detection of myoglobin over a wide range of concentrations from subnormal to the elevated level of this cardiac marker. Utilizing SPCE allowed us also to demonstrate significant background suppression (from serum or whole blood) in the myoglobin immunoassay. We expect SPCE to become a powerful technique for performing immunoassays for many biomarkers in surface-bound assays.

First observation of surface plasmon-coupled chemiluminescence (SPCC)

“…It is however worth noting that the signal at the SPCC peak angle is not entirely ppolarized. This is in contrast with our past experiences with optically pumped SPCE experiments where the SPCE signal was almost entirely p-polarized [8][9][15][16][17][18][19][20]. Figure 2 (Bottom) is the normalized SPCC and free-space emission spectra showing a high degree of overlap between the spectra.…”

“…This is unlike our previous experiences with optically pumped SPCE studies for a thin layer of fluorophores, where the SPCE peak angle was approximately 1-2 degrees [8][9][15][16][17][18][19][20]. In order to investigate if the broadness of the SPCC peak is dependent on the surface area of the sample, we repeated the experiment with a sample chamber built inhouse that had approximately half the surface area when compared to the samples made with commercially available imaging chambers that had been used thus far.…”

“…However, the hydrophobic nature of the surface globulated the chemiluminescence liquid, preventing films less < 250 nm thick to be produced. Given that clinical/biochemical assays are typically substantially smaller than 250 nm, indeed as we have shown in numerous SPCE publications [15][16][17][18][19][20], then similarly we expect surface-bound chemiluminescence assays to be also highly useful. Figure 2 (Top) is an enlarged figure showing the highly directional and p-polarized SPCC emission, suggesting that the observed signal is due to surface plasmons.…”

“…We believe this phenomenon is not restricted to the commercially available kits that were used in this study, but moreover, can be extended to the myriad of chemiluminescent reactions employed in biotechnology today to increase signal collection efficiency and hence the sensitivity of such assays. Interestingly, the physical dimensions of these assays are compatible with an approximately 250 nm coupling region [15][16][17][18][19][20], potentially alleviating unwanted background distal fluorescence and therefore increasing assay sensitivity. In this regard, work is underway in our laboratories and will be reported in due course.…”

“…We subsequently termed this phenomenon as surface plasmon-coupled emission (SPCE) [8][9][15][16][17][18][19][20]. We have also shown that SPCE can be generated on gold films by electrochemical excitation of [Ru(bpy) 3 ] 2+ near a gold electrode [21].…”