Surface enhanced Raman scattering (SERS) is an extremely sensitive and selective spectroscopic technique commonly employed for trace environmental analyses. We are currently developing and optimizing novel SERS based immuno-nanosensors for the monitoring of protein expression within individual living cells. These sensors, based upon antibody bound, silver coated silica nanospheres, can be inserted into individual cells and non-invasively positioned to the subcellular location of interest using optical tweezers. Because a HeNe laser is used for excitation, both photodamage to cells and cellular autofluorescence are minimized.
Fabrication of the nanosensors is performed by first depositing a thin layer of silver on silica nanospheres by either homogeneous chemical deposition or vapor deposition. This is followed by binding an antibody against the analyte of interest to the sensor’s surface. In this paper we have investigated and optimized the reduction conditions of a chemical deposition substrate fabrication method and have compared these substrates to three types of vapor deposited substrates: (1) single layer silver film over nanosphere (SFON), (2) dual layer silver film over nanosphere (DUAL-FON) substrates (produced by coating an additional layer of silver film over the SFON), and (3) multilayer silver film over nanosphere (MULTI-FON) substrates (prepared by repeated coatings of several silver films over a SFON). In the case of chemical deposition, parameters optimized included silver nitrate concentration, reaction temperature, and silver coating time of substrates. In general, we have found SFON substrates yield better signal-to-noise ratios (S/N) with less background than the optimized, chemically prepared substrates. Even greater SERS enhancements have been found using multilayered SERS substrates (i.e., DUAL-FON substrates and MULTI-FON substrates). Both the stability and S/N of these substrates are enhanced by using multiple silver layers compared to SFON substrates of similar thickness. From this work, SERS signal enhancements of over an order of magnitude can be predicted by combining the enhancements obtained from the multilayered geometry and that from optimized silver deposition parameters.
This paper includes investigations into the mechanism of multi-layer enhancement by studying the SERS S/N changes after different amount of exposure time of underlayer silver films to air. From these studies, we suggest that silver oxide layers play an important role in this multilayered enhancement. To the best of our knowledge, this paper is the first report of this type of silver oxide layer production affecting and even enhancing the resulting SERS signals from continuous film SERS substrates. In this paper, the fabrication, characterization and comparison of these SERS substrates in terms of enhancement factor, and lifetime will be discussed.