A Nanoscale Optical Biosensor:  Real-Time Immunoassay in Physiological Buffer Enabled by Improved Nanoparticle Adhesion

The shift in the extinction maximum, λmax, of the localized surface plasmon resonance (LSPR) spectrum of triangular Ag nanoparticles (∼90 nm wide and 50 nm high) is used to probe the interaction between a surface-confined antigen, biotin (B), and a solution-phase antibody, anti-biotin (AB). Exposure of biotin-functionalized Ag nanotriangles to 7 × 10-7 M < [AB] < 7 × 10-6 M caused a ∼38 nm red-shift in the LSPR λmax. The experimental normalized response of the LSPR λmax shift, (ΔR/ΔR max), versus [AB] was measured over the concentration range 7 × 10-10 M < [AB] < 7 × 10-6 M. Comparison of the experimental data with the theoretical normalized response for a 1:1 binding model yielded values for the saturation response, ΔR max = 38.0 nm, the surface-confined thermodynamic binding constant, K a,surf = 4.5 × 107 M-1, and the limit of detection (LOD) < 7 × 10-10 M. The experimental saturation response was interpreted in terms of a closest-packed structural model for the surface B−AB complex in which the long axis of AB, l AB = 15 nm, is oriented horizontally and the short axis, h AB = 4 nm is oriented vertically to the nanoparticle surface. This model yields a quantitative response for the saturation response, ΔR max = 40.6 nm, in good agreement with experiment, ΔR max = 38.0 nm. An atomic force microscopy (AFM) study supports this interpretation. In addition, major improvements in the LSPR nanobiosensor are reported. The LSPR nanobiosensor substrate was changed from glass to mica, and a surfactant, Triton X-100, was used in the nanosphere lithography fabrication procedure. These changes increased the adhesion of the Ag nanotriangles by a factor of 9 as determined by AFM normal force studies. The improved adhesion of Ag nanotriangles now enables the study of the B−AB immunoassay in a physiologically relevant fluid environment as well as in real-time. These results represent important new steps in the development of the LSPR nanosensor for applications in medical diagnostics, biomedical research, and environmental science.