Joseph R. Lakowicz

2 – Basics of Fluorescence and FRET

This chapter discusses the most important characteristics of fluorescence that plays a fundamental role in understanding the basics and the applications of Forster (fluorescence) resonance (radiationless) energy transfer (FRET). FRET is the transfer of electronic excitation energy between isolated donor D and acceptor A of suitable spectroscopic properties. The donor molecules, typically, emit at shorter wavelengths, which overlap with the absorption spectrum of the acceptor. This energy transfer occurs without the appearance of the photon and is the result of long-range interactions between the D and A dipoles. The most important factors affecting FRET are the overlap integral, the quantum yield of the donor in the absence of the acceptor, and the orientation factor. The quantitative analysis of steady-state and time-resolved FRET measurements provides information on global structures and conformational dynamics, and reveals thermodynamic parameters for conformational transition. This information is essential for the understanding of biological functions of proteins, DNA/RNA, and other biological assemblies that are frequently mediated by transitions between alternative conformations.

Fractal Silver Structures for Metal-Enhanced Fluorescence: Applications for Ultra-Bright Surface Assays and Lab-on-a-Chip-Based Nanotechnologies

119 1053-0509/03/0300-0119/0

Noble-Metal Surfaces for Metal-Enhanced Fluorescence

Noble metal nanoparticles exhibit strong absorption bands, which known as the surface plasmon resonances, result in strong absorption and scattering, and create an enhanced local electromagnetic field near-to the surface of the particles. The surface plasmon resonances are highly dependent on the size and the shape of the metal and the dielectric properties of the surrounding medium. These near field enhancements have given rise to surface-enhanced resonant Raman scattering (SERRS) and metal-enhanced fluorescence (MEF) (Figure 1). Unlike SEERS, the optimal MEF signal occurs at a certain distance from the surface of the metal nanoparticles. The fluorophores in direct contact with the metal surface are typically quenched. Theoretical and experimental work using rough surfaces and particles has suggested that the distance-dependent enhancement fluorescence intensity is more pronounced for low quantum yield fluorophores.1–8 This enhancement is accompanied by a significantly reduced lifetime. The increased fluorescence intensities accompanied by reduced lifetimes suggest an increased radiative decay rate for the fluorophores interacting with the metals.

Distance-dependent intrinsic fluorescence of proteins on aluminum nanostructures

In the past several years we have demonstrated the metal-enhanced fluorescence (MEF) and the significant changes in the photophysical properties of fluorophores in the presence of metallic nanostructures and nanoparticles using ensemble spectroscopic studies. In the represented study, we explored the distance effect on intrinsic fluorescence of proteins adsorbed on our layer-by-layer assembled metallic nanostructures. The study is expected to provide more information on the importance of positioning the proteins at a particular distance for enhanced fluorescence from metallic structures. For the present study, we considered using easy and inexpensive LbL technique to provide welldefined distance from metallic surface. The explored proteins were adsorbed on different numbers of alternate layers of poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH). SA and BSA were electrostatically attached to the positively charged PAH layer. We obtained a maximum of ~11-fold and 9-fold increase in fluorescence intensity from SA and BSA, respectively. And also we observed ~3-fold decrease in BSA lifetime on metallic nanostructures than those on bare control quartz slides. This study reveals the distance dependence of protein fluorescence.

Fluorescence enhancement using silver-gold nanocomposite substrates.

Metal-enhanced fluorescence (MEF) is a newly emerging phenomenon in which the near-field interactions of fluorophores with the plasmons in metallic nanostructures can lead to substantial fluorescence enhancements. In the present study, we have investigated the use of silver-gold nanocomposite (Ag-Au-NC) structures, prepared by the galvanic replacement reaction of silver with gold, as plasmonic substrates for MEF. We have observed significant enhancement in the fluorescence intensities and decrease in the fluorescence lifetimes of two commonly used dyes, ATTO655 and Cy5, using the fabricated Ag-Au-NC substrates. Interestingly, the fluorescence enhancement depends on the amount of residual silver present in the substrates after the galvanic replacement reaction. Our results show that the galvanic replacement reaction is a very facile and powerful route to prepare Ag-Au-NC substrates that can be suitable for various MEF based applications.

Plasmon-controlled fluorescence and single DNA strand sequenching

During the past several years we have studied the effects of metallic surfaces and nanostructures with fluorophores. We have demonstrated the metal-enhanced fluorescence (MEF) and the significant changes in the photophysical properties of fluorophores in the presence of metallic nanostructures and nanoparticles using ensemble spectroscopic studies. These studies have shown dramatic increases in brightness and photostability, especially for low quantum yield fluorophores. Much of this work was performed using visible or NIR fluorophores. In the present study, we have extended our studies to UV wavelengths and have shown that aluminum and platinum particles can enhance the emission of UV fluorophores including intrinsic protein fluorescence from 300 to 420 nm. We used the finite-difference timedomain (FDTD) method to calculate the effects of aluminum nanoparticles on nearby fluorophores that emit in the UV. And also we performed experiments to investigate the effect of metallic nanoparticles on fluorescence intensity of DNA bases and DNA G-quadruplex. We observed increase in fluorescence intensities of DNA bases varied range changing from 20 to 3-fold in steady-state fluorescence emission measurements. We obtained ~5-fold increase in fluorescence intensity of DNA G-quadruplex on both Al and Pt metallic substrates when compared with control quartz substrates.