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.

Study of the Aggregation of DNA‐Capped Gold Nanoparticles: A Smart and Flexible Aptasensor for Spermine Sensing

“…As revealed from the absorption pattern, with a higher SPM concentration, the plasmonic peak declined gradually, similar to the previous titration study of the artificial urine matrix effect. This confirmed that SPM could also help to screened negative charges such as salts and induced further DNA adsorption . Therefore, a greater extent of DNA adsorption or even AuNP precipitation was observed upon addition of SPM (Figure S14 in the Supporting Information).…”

Surface-enhanced Raman spectroscopy for bioanalysis and diagnosis

“…19 SERS label-based detection aims to recognize the vibrational spectroscopic fingerprints of bioanalytes via indirect interaction, by using Raman reporter molecules to label the SERS tags; for instance, this detection is widely used in SERS-based DNA sensing. [20][21][22] Label-free SERS detection is a direct approach; it senses and images the bioanalytes after adsorption on the SERS substrates/nanostructures, which often results in improved signal intensity. 23 In the label-based detection scheme, the SERS nano-substrates comprise four distinct parts: (1) a nanostructured metallic substrate, usually made of gold or silver to improve the SERS intensity and activity; (2) an appropriate protective shell or layering to enhance the stability and biocompatibility; (3) a Raman reporter molecule layer to enable labelbased detection with a unique SERS fingerprint; and (4) targetspecific detection using bioconjugations.…”

Fundamentals and applications of SERS-based bioanalytical sensing

“…The sandwich approach uses target DNA-Raman reporter molecules [225,[230][231][232][233][234][235][236][237][238][239][240]. The first report of SERS DNA and RNA detection using sandwich structures was published by Mirkin and co-workers.…”