Plasmonic Enhancement of Two-Photon Excitation Fluorescence by Colloidal Assemblies of Very Small AuNPs Templated on M13 Phage.

In this study, an engineered M13 bacteriophage was examined as a biological template to create a well-defined spacing between very small gold nanoparticles (AuNPs 3-13 nm). The effect of the AuNP particle size on the enhancement of the nonlinear process of two-photon excitation fluorescence (2PEF) was investigated. Compared to conventional (one-photon) microscopy techniques, such nonlinear processes are less susceptible to scattering given that the density of background-scattered photons is too low to generate a detectable signal. Besides this, the use of very small AuNPs in 2PEF microscopy becomes more advantageous because individual "isolated" AuNPs of this size do not sufficiently enhance 2PEF to produce a detectable signal, resulting in even less background signal. To investigate the 2PEF of the AuNP-M13 assemblies, a variety of sample preparation approaches are tested, and surface-enhanced Raman spectroscopy (SERS) is employed to study the strength of plasmon coupling within the gaps of AuNPs assembled on the M13 template. Results indicate that assemblies prepared with 9-13 nm AuNP were able to clearly label cells and produce a 2PEF signal that was orders of magnitude higher than the isolated AuNP (below the threshold of detection). This study thus provides a better understanding of the opportunities and limitations relevant to the use of such small AuNPs within colloidal plasmonic assemblies, for applications in biodetection or as imaging contrast agents.