title: Simultaneous detection of transgenic DNA by surface plasmon resonance imaging with potential application to gene doping detection.
abstract: Surface plasmon resonance imaging (SPRi) was used as the transduction principle for the development of optical-based sensing for transgenes detection in human cell lines. The objective was to develop a multianalyte, label-free, and real-time approach for DNA sequences that are identified as markers of transgenosis events. The strategy exploits SPRi sensing to detect the transgenic event by targeting selected marker sequences, which are present on shuttle vector backbone used to carry out the transfection of human embryonic kidney (HEK) cell lines. Here, we identified DNA sequences belonging to the Cytomegalovirus promoter and the Enhanced Green Fluorescent Protein gene. System development is discussed in terms of probe efficiency and influence of secondary structures on biorecognition reaction on sensor; moreover, optimization of PCR samples pretreatment was carried out to allow hybridization on biosensor, together with an approach to increase SPRi signals by in situ mass enhancement. Real-time PCR was also employed as reference technique for marker sequences detection on human HEK cells. We can foresee that the developed system may have potential applications in the field of antidoping research focused on the so-called gene doping.
authors.affiliation: Dipartimento di Chimica, Università degli Studi di Firenze Ugo Schiff, Sesto Fiorentino, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Scarano
authors.forename: Maria Laura
authors.initials: ML
authors.lastname: Ermini
authors.forename: Maria Michela
authors.initials: MM
authors.lastname: Spiriti
authors.forename: Marco
authors.initials: M
authors.lastname: Mascini
authors.forename: Patrizia
authors.initials: P
authors.lastname: Bogani
authors.forename: Maria
authors.initials: M
authors.lastname: Minunni
mesh_terms: D001483:Base Sequence; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods; D001710:Biotin / Q000378:metabolism; D004247:DNA / Q000032:analysis*; D015342:DNA Probes / Q000138:chemical synthesis / Q000378:metabolism; D004300:Doping in Sports / Q000517:prevention & control*; D006046:Gold / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000032:analysis; D006801:Humans; D056656:Lab-On-A-Chip Devices; D009693:Nucleic Acid Hybridization / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction; D013194:Staining and Labeling / Q000379:methods*; D019809:Streptavidin / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods; D019076:Transgenes
chemical_list: D015342:DNA Probes; D013438:Sulfhydryl Compounds; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D001710:Biotin; D006046:Gold; D004247:DNA; D019809:Streptavidin