The quenching of electrochemiluminescence upon oligonucleotide hybridization

Abstract

Many genomic assays rely on a distance-dependent interaction between luminescent labels, such as luminescence quenching or resonance energy transfer. We studied the interaction between electrochemically excited Ru(bpy)(3) (2+) and Cy5 in a hybridization assay on a chip. The 3' end of an oligonucleotide was labelled with Ru(bpy)(3) (2+) and the 5' end of a complementary strand with Cy5. Upon the hybridization, the electrochemiluminescence (ECL) of Ru(bpy)(3) (2+) was efficiently quenched by Cy5 with a sensitivity down to 30 nmol/L of the Cy5-labelled complementary strand. The quenching efficiency is calculated to be 78%. A similar phenomenon was observed in a comparative study using laser-excitation of Ru(bpy)(3) (2+). The hybridization with the non-labelled complementary or labelled non-complementary strand did not change the intensity of the ECL signal. Resonance energy transfer, electron transfer and static quenching mechanisms are discussed. Our results suggest that static quenching and/or electron transfer are the most likely quenching mechanisms.

Electrochemiluminescent hybridization chip with electric field aided mismatch discrimination

“…The labeling efficiency was calculated to be 100% using absorption of oligonucleotides at 260 nm and of ruthenium complex at 455 nm. The cyclic voltammograms were recorded using Autolab PGSTAT12 (Eco chemi) potentiostat in a three-electrode mode against a silver pseudoreference electrode on chip made by placing a droplet of silver conductive glue on a gold electrode (Spehar et al, 2004). Cyclic voltammograms were measured in 10 mM tris buffer, pH 7.0 sweeping potential first in negative direction, and back to positive.…”

“…Based on previous experience, we first measured the ECL signal in a 300 mM phosphate buffer containing 100 mM TPA, pH 7.8 (Spehar et al, 2004). However, the intensity of the ECL was very low (Fig.…”

“…Wong et al, 2005), microgravimetrical (Su et al, 2004), bioluminescence (Kricka, 1999), chemiluminescence (Cheek et al, 2001) and electrochemiluminescence (ECL) (Miao and Bard, 2003;Spehar et al, 2004;Bertolino et al, 2005;Firrao, 2005) methods have been used.…”

Heterogeneous oligonucleotide-hybridization assay based on hot electron-induced electrochemiluminescence of a rhodamine label at oxide-coated aluminum and silicon electrodes

“…Of particular interest is the detection of DNA strands of specific sequence due to applications in forensic science, food analysis, pharmacology, genomics, etc. Most DNA hybridization assays rely on fluorescence detection, but other methods, such as quartz crystal microbalance [1], electrochemistry [2,3], chemiluminescence [4] and anodic electrochemiluminescence (ECL) [5][6][7][8][9] have also been applied.…”

“…In anodic ECL, an electrochemiluminescent luminophore (usually tris(bipyridine)ruthenium, Ru(bpy) 3 2+ ) is oxidized on a surface of a metal or carbon electrode and light is generated upon reaction with a coreactant (typically ntripropylamine) [5][6][7][8][9].…”