Two-round enzymatic amplification combined with time-resolved fluorometry of Tb3+ chelates for enhanced sensitivity in DNA hybridization assays.

Microtiter well-based DNA hybridization assays are developed in which two rounds of enzymatic amplification are combined with time-resolved fluorometry of Tb3+ chelates for enhanced sensitivity. The target DNA is immobilized on the wells (through digoxigenin/anti-digoxigenin interaction) and then hybridized with a biotinylated oligonucleotide probe. The hybrids are reacted with a streptavidin-horseradish peroxidase conjugate. Peroxidase catalyzes the oxidation of biotinylated tyramine by hydrogen peroxide, resulting in the attachment of multiple biotin moieties to the solid phase. Alkaline phosphatase-labeled streptavidin is then allowed to bind to the immobilized biotins. The activity of alkaline phosphatase is measured by using the phosphate ester of 5'-fluorosalicylate as a substrate. The fluorosalicylate produced forms a fluorescent complex with Tb3+, which is measured by time-resolved fluorometry. We observed a 30-fold improvement of the signal and a 10-times enhancement of the signal-to-background ratio compared to the assay that uses a single round of enzymatic amplification (only alkaline phosphatase). The CV was in the range of 11.2-14.4%.