Metal-Enhanced Intrinsic Fluorescence of Proteins on Silver Nanostructured Surfaces toward Label-Free Detection

In recent years, metal-enhanced fluorescence (MEF) using silver particles has been reported for a number of fluorophores emitting at visible wavelengths. However, it was generally thought that silver particles would always quench fluorescence at shorter wavelengths. We now report the observation of MEF of the tryptophan analogue N-acetyl-L-tryptophanamide (NATA) on silver nanostructured surfaces. NATA is a model for the intrinsic tryptophan emission from proteins. We have also studied the effects of silver nanostructures on the emission of N-acetyl-L-tyrosinamide (NATA-tyr). In the case of NATA, we observed increased emission, decrease in fluorescence lifetimes, and increase in photostability when NATA was embedded in 15 nm thick spin-casted poly(vinyl alcohol) film on silver nanostructured surfaces. We have also investigated the effects of silver nanostructures on the emission from thin poly(vinyl alcohol) films containing NATA-tyr. However, we observed no increase in fluorescence signal for NATA-tyr on silver nanostructures. To understand these results, we performed numerical calculations using the finite-difference time-domain (FDTD) technique to model a tryptophan-wavelength dipole near a spherical silver particle. Our calculations reveal an enhancement of the power of the radiated emission by the excited-state fluorophore in proximity to a 100 nm diameter silver nanoparticle covering the emission spectra of NATA and NATA-tyr. These calculations show a clear wavelength dependence with the specific spectral region displaying low-enhancement at the shorter NATA-tyr wavelength and higher enhancement at NATA emission wavelength. Our FDTD calculations also reveal that excited fluorophores in the near-field of a 100 nm silver nanoparticle can induce enhancement fields of varying degrees of the intensity of the near-fields around the particle that is dependent on the wavelength of the emission. We believe these enhanced near-fields play a role in our observation of MEF from metal surfaces. The enhanced emission of NATA on silver nanostructures suggests that the extension of MEF to the UV region opens new possibilities to study tryptophan-containing proteins without labeling with longer wavelength fluorophores, leading to label-free detection of biomolecules.