title: Preferential Attachment of Specific Fluorescent Dyes and Dye Labeled DNA Sequences in a Surface Enhanced Raman Scattering Multiplex.
abstract: 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.
authors.affiliation: Centre of Molecular Nanometrology, Technology Innovation Centre, University of Strathclyde , 99 George Street, Glasgow, G1 1RD, United Kingdom.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Gracie
authors.affiliation: Department of Statistics, University of Warwick , Coventry, CV4 7AL, United Kingdom.
authors.forename: Matthew
authors.initials: M
authors.lastname: Moores
authors.affiliation: Centre of Molecular Nanometrology, Technology Innovation Centre, University of Strathclyde , 99 George Street, Glasgow, G1 1RD, United Kingdom.
authors.forename: W Ewen
authors.initials: WE
authors.lastname: Smith
authors.affiliation: Centre of Molecular Nanometrology, Technology Innovation Centre, University of Strathclyde , 99 George Street, Glasgow, G1 1RD, United Kingdom.
authors.forename: Kerry
authors.initials: K
authors.lastname: Harding
authors.affiliation: Department of Statistics, University of Warwick , Coventry, CV4 7AL, United Kingdom.
authors.forename: Mark
authors.initials: M
authors.lastname: Girolami
authors.affiliation: Centre of Molecular Nanometrology, Technology Innovation Centre, University of Strathclyde , 99 George Street, Glasgow, G1 1RD, United Kingdom.
authors.forename: Duncan
authors.initials: D
authors.lastname: Graham
authors.affiliation: Centre of Molecular Nanometrology, Technology Innovation Centre, University of Strathclyde , 99 George Street, Glasgow, G1 1RD, United Kingdom.
authors.forename: Karen
authors.initials: K
authors.lastname: Faulds
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D019793:Fluorescein / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D015394:Molecular Structure; D012235:Rhodamines / Q000737:chemistry*; D013059:Spectrum Analysis, Raman / Q000379:methods*; D013499:Surface Properties
chemical_list: D005456:Fluorescent Dyes; D012235:Rhodamines; C005358:tetramethylrhodamine; D004247:DNA; D019793:Fluorescein