title: Direct detection of genomic DNA by surface plasmon resonance imaging: an optimized approach.
abstract: The direct detection of specific sequences in genomic DNA samples is very challenging in the biosensor-based approach. In this work we developed an optimized strategy for the direct detection of DNA sequences in human genomic samples by a surface plasmon resonance imaging technology. As model study, the target analyte was identified in a DNA sequence mapping the human ABCB1 gene. The computed-assisted approach was here applied for probe design. After a preliminary evaluation of the probe functioning by the complementary synthetic target, the system was applied to the direct detection of the target sequence in human genomic DNA extracted from lymphocytes. To achieve this result, several steps aimed to improve the analytical performances of the biosensor were studied and optimized. The immobilization chemistry, based on thiolated probes, was adapted here to non-amplified sequence detection. DNA sample pre-treatments, i.e. genomic fragmentation by ultrasounds and dsDNA denaturation by thermal treatment were also investigated. A sandwich-like strategy, by using a secondary probe, was also applied to understand and confirm the selectivity of the developed biosensor in detecting ABCB1 gene in genomic samples. Finally, a reliable calibration curve of ABCB1 was obtained with an experimental detection limit of 140 aM. Furthermore, the biosensor was well regenerable, assuring up to thirty cycles of effective measurements.
authors.affiliation: Dipartimento di Chimica Ugo Schiff and CSGI, Università degli Studi di Firenze,Via della Lastruccia 3, 50019 Sesto F.no (FI), Italy.
authors.forename: M L
authors.initials: ML
authors.lastname: Ermini
authors.forename: S
authors.initials: S
authors.lastname: Mariani
authors.forename: S
authors.initials: S
authors.lastname: Scarano
authors.forename: M
authors.initials: M
authors.lastname: Minunni
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation*; D002874:Chromosome Mapping / Q000379:methods*; D004247:DNA / Q000032:analysis / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance / Q000295:instrumentation*
chemical_list: D004247:DNA