A significant advantage of using surface enhanced Raman scattering (SERS) for DNA detection is the capability to detect multiple analytes simultaneously within the one sample. However, as the analytes approach the metallic surface required for SERS, they become more concentrated and previous studies have suggested that different dye labels will have different affinities for the metal surface. Here, the interaction of single stranded DNA labeled with either fluorescein (FAM) or tetramethylrhodamine (TAMRA) with a metal surface, using spermine induced aggregated silver nanoparticles as the SERS substrate, is investigated by analyzing the labels separately and in mixtures. Comparison studies were also undertaken using the dyes in their free isothiocyanate forms, fluorescein isothiocyanate (F-ITC) and tetramethylrhodamine isothiocyanate (TR-ITC). When the two dyes are premixed prior to the addition of nanoparticles, TAMRA exerts a strong masking effect over FAM due to a stronger affinity for the metal surface. When parameters such as order of analyte addition, analysis time, and analyte concentration are investigated, the masking effect of TAMRA is still observed but the extent changes depending on the experimental parameters. By using bootstrap estimation of changes in SERS peak intensity, a greater insight has been achieved into the surface affinity of the two dyes as well as how they interact with each other. It has been shown that the order of addition of the analytes is important and that specific dye related interactions occur, which could greatly affect the observed SERS spectra. SERS has been used successfully for the simultaneous detection of several analytes; however, this work has highlighted the significant factors that must be taken into consideration when planning a multiple analyte assay.

a b s t r a c t O 1s inner-shell excitation spectra of a number of vapor phase molecules containing peroxide bonds -hydrogen peroxide (H 2 O 2 ), di-t-butylperoxide ( t BuO t Bu), benzoyl peroxide, ((C 6 H 5 (CO)O) 2 ), luperox-F [1,3(4)-bis(tertbutylperoxyisopropyl) benzene], and analogous, non-peroxide compounds -water, t-butanol and benzoic acid have been measured. C 1s spectra are also reported. O 1s spectra of solid benzoic acid, di-t-butylperoxide and luperox-F recorded using a scanning transmission X-ray microscope, are also reported, and compared to the corresponding gaseous spectra. Spectral interpretation was aided by comparing the spectra of the peroxide and non-peroxide counterparts and with ab initio calculations. A characteristic O 1s ? r ⁄ O-O transition at 533.0(3) eV is identified in each peroxide species, which is absent in the corresponding non-peroxide counterpart species. The energy and intensity of the 533 eV peroxide feature is stable and thus useful for analysis of peroxides in mixtures, such as tracking residual peroxide initiators, or peroxides produced in fuel cells.

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