Dual signal amplification for highly sensitive hybridization microassays on chemically activated surfaces

Abstract

[EN] A highly sensitive hybridization assay is developed for detection of specific sequences of genomic DNAwithout thermal or isothermal chain reaction cycling. The assays in microarray format were performedon polymeric surfaces derived from the audio-video disc industry. In order to achieve this challenge, aphoto-chemical activation method for the covalent immobilization of DNA oligomers is developed. Inaddition, dual signal amplification approach, by integrating tyramide with silver-assisted enhancementfor the detection of DNA at low pg/L level, is presented.The methodology was two orders of magnitude more sensitive in comparison with the non-tyramideamplified hybridization assay. To demonstrate the feasibility of this approach, complementary oligomersand PCR products as a model targets were employed, achieving a quantification limit of 0.30 pM and0.2 pg/L, respectively. The reproducibility was good (RSD <18%) and the hybridization assay took 65 min.As a proof of concept, the detection of genomic DNA of Salmonella spp., as food-borne pathogen model,was addressed. The principle of assay is based on massive biotinylation and fragmentation of genomicDNA prior the hybridization step. The biotinylated fragments, ranging from 400 to 750 bp hybridize withthe covalently immobilized specific probes (38 mer long), generating an analytical signal after tyramideamplification proportional to the concentration of DNA. The signal is quantified with a disc drive asdetector. The results showed that the methodology is a promising alternative for DNA sensing withoutthermal or isothermal cycling based amplification.