Spacer length, label moiety interchange and probe pair orientation in a homogeneous solid-phase hybridization assay utilizing lanthanide chelate complementation

Abstract

We have studied parameters affecting DNA hybridization and lanthanide chelate complementation based signal formation in a separation-free solid-phase assay suitable for spatial multiplexing. This binary probe assay system consists of two probes labeled either with a europium carrier chelate or a light harvesting antenna ligand. One probe was immobilized on the microtiter well bottom in a spot format while the other probe was free in solution. The probe concentration used in spotting, spacer length, and the choice and orientation of the either 3′- or 5′-end immobilized probe had a significant impact on signal-to-background (S/B) ratios. The highest ratio was achieved by saturating the spot with the 5′-end immobilized antenna ligand probe separated from the solid support with a 25 nucleotide poly dT spacer. The dynamic range of the assay for the synthetic Pseudomonas aeruginosa heat shock protein groES gene target was three orders of magnitude and the detection limit of 18 pM was obtained. The groES target sequence was also amplified from isolated genomic DNA of P. aeruginosa both with and without co-amplification of synthetic internal assay control (IAC) followed by successful detection of the groES amplification product although the co-amplification of IAC resulted in a slightly decreased groES signal. The homogeneous detection of spatially resolved probes on a solid-phase could enable an alternative way of multiplexing to closed tube nucleic acid assays.