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.

Figure 1.

Top: Experimental geometry for measurements of SPCE emission with RK and KR configurations. In RK configuration, the sample is excited directly by 532 nm and the emission out-couples through the prism in a hollow cone at the angle θ_F. In KR configuration, the excitation is provided by the evanescent field created by the incident light of 532 nm, which enters the system through the prism at the angle of θ_I. The fluorophores excited by this evanescent field couple to the surface plasmons, and the directional emission out-couples through the prism at the angle θ_F. The emission is collected by the fiber equipped with the filter (F) and polarizer (P). Bottom: Scheme of the myoglobin immunoassay (sandwich format) on a thin silver mirror slide surface. The drawing is not to the scale. The thickness of the silver layer was 50 nm and SiO₂ protective layer 5 nm.

Figure 2.

Polarized fluorescence spectra of the Rhodamine Red-X-labeled anti-myoglobin antibodies bound to the captured myoglobin observed at 72° in RK/SPCE configuration (see Figure 1, top).

Figure 3.

Angular distribution of the 590-nm fluorescence emission of Rhodamine Red-X-labeled anti-myoglobin antibodies bound to the myoglobin captured on the 50-nm silver mirror surface. The measurements were done in KR configuration with the excitation angle θ_I = 74.5°.

Figure 4.

Calculated reflectivity curves for a 50-nm silver film on BK7 glass (n_p = 1.52). The sample (protein layers) was assumed to be 18 nm thick (n_s = 1.50). The buffer thickness was taken as infinite with n_w = 1.33. For silver phase we used ϵ_m⁵³² = −11.5 + 0.3i, and ϵ_m⁵⁹⁰ −15.0 + 0.4i.

Figure 5.

Top: Kinetics of binding of the Rhodamine Red-X-labeled anti-myoglobin antibodies to myoglobin (0–1000 ng/mL) captured on the 50-nm silver mirror surface observed with KR/SPCE configuration: A, noncorrected data; B, data after subtraction of background. Bottom: Dependence of SPCE signal (after 15 min) on the Myo concentration. Dashed line represents the log trend (linear at log Myo concentration scale).

Figure 6.

Fluorescence spectra of the Rhodamine Red-X-labeled anti-myoglobin antibodies bound to the captured myoglobin (at [Myo] = 50 ng/mL) observed with KR/SPCE configuration: 1, in blocking buffer solution; 2, in human serum solution; 3, in 17% bovine hemoglobin solution.