3.3.1 Introduction

Abstract

Techniques that use fluorescence probes based on fluorescence energy transfer (e.g., molecular beacons) have been widely applied with great success in biosensing assays. Nevertheless, the need for alternative, rapid, and selective assays has contin-ued to encourage researchers to explore other technologies hav-ing comparable sensitivity as fluorescence but having additional unique and complementary advantages. Raman spectroscopy is an important analytical technique for chemical and bio-logical analysis due to the wealth of information on molecular structures, surface processes, and interface reactions that can be extracted from experimental data. The spectral selectivity associated with the narrow emission lines and the molecular-specific vibrational bands of Raman labels make it an ideal tool for molecular genotyping. However, a limitation of Raman tech-niques for trace detection is the very weak Raman cross section. However, Raman spectroscopy has gained increasing interest as an analytical tool with the advent of the surface-enhanced Raman scattering (SERS) and surface-enhanced resonance Raman scattering (SERRS) effects, which can produce signifi-cant enhancement of the Raman signal. It is believed that the origin of the enormous Raman enhancement is produced by at least two main mechanisms that contribute to the SERS effect: (a) an electromagnetic effect occurring near metal surface struc-tures associated with large local fields caused by electromag-netic resonances, often referred to as " surface plasmons " and (b) a chemical effect involving a scattering process associated with chemical interactions between the molecule and the metal sur-face. Plasmons are quanta associated with longitudinal waves propagating in matter through the collective motion of large numbers of electrons. According to classical electromagnetic theory, molecules on or near metal nanostructures experience enhanced fields relative to that of the incident radiation. When a metallic nanostructured surface is irradiated by an incident electromagnetic field (e.g., a laser beam), conduction electrons are displaced into frequency oscillations equal to those of the incident light. These oscillating electrons, called " surface plas-mons, " produce a secondary electric field, which adds to the incident field. These fields can be quite large (10 6 –10 7 -, even up to 10 15 -fold enhancement at " hot spots "). When these oscillat-ing electrons become spatially confined, as is the case for iso-lated metallic nanospheres or otherwise roughened metallic surfaces (nanostructures), there is a characteristic frequency (the plasmon frequency) at which there is a resonant response of the collective oscillations to the incident field. This condition yields intense localized fields that can interact with molecules in contact with or near the metal surface (Otto 1978, Gersten and Nitzan 1980, Schatz 1984, Zeman and Schatz 1987). In an effect analogous to a " lightning rod " effect, secondary fields can become concentrated at high curvature points on the roughened metal surface. In SERRS, the energy of the incoming laser is selected such that it coincides with an electronic transition of the molecule being monitored. An advantage of SERRS over SERS is the large increase in intensity of the Raman peaks. Following the discovery of the SERS effect (Fleischmann et al. 1974, Albrecht and Creighton 1977, Jeanmaire and Vanduyne 1977), our laboratory has first demonstrated the general applica-bility of the SERS effect for trace analysis using solid substrates having silver-coated nanospheres (Vo-Dinh et al. 1984). In 1984, 33