Magnetic bead-based chemiluminescence detection of sequence-specific DNA by using catalytic nucleic acid labels

We wish to report a new chemiluminescence (CL) method for the determination of the sequence-specific DNA by the coupling of catalytic nucleic acid label (DNAzyme) based CL detection route with an efficient magnetic isolation of the hybrid. The assay relies on (i) the immobilization of NH2-modified capture DNA on the surface of carboxyl-terminated magnetic beads activated by EDC, (ii) first hybridization event occurring between the bead-captured DNA probe and one 15-mer portion (5′-AAT ATT GAT AAG GAT-3′) of the target sequence, (iii) second hybridization happening between one part of the DNAzyme modified reporter sequence and another 15-mer portion (5′-GAG GGA TTA TTG TTA-3′) of the target sequence, and then direct detection of CL signal on the surface of the magnetic beads in a basic solution of luminol and H2O2. The influence of relevant experimental variables, including the effect of the amount of the magnetic beads, the duration of the hybridization and the parameters affecting CL signal, is examined and optimized. The results showed that this simple and sensitive protocol was suitable for the determination of specific-sequence DNA as exemplified by the 30-base sequence related to the anthrax lethal factor with a good linear correlation in the concentration range of 0.02–2 pmol (R2 = 0.9987) and a detection limit of 1 × 10−10 M. Overall, this new CL protocol coupled the high sensitivity of CL analysis with effective magnetic separation for discriminating against unwanted constituents such as mismatched sequences and proteins, hence, offers great promise for DNA hybridization analysis.