Peptide nucleic acid modified magnetic beads for intercalator based electrochemical detection of DNA hybridization

The specific binding of peptide nucleic acid (PNA) to its complementary DNA target is combined with magnetic separation to enable discrimination against single nucleotide polymorphisms (SNP). PNA probes with biotin label at 5'-end were attached to strepavidin coated superparamagnetic iron oxide beads. PNA modified beads were then challenged with non-complementary, SNP containing and perfect-match DNA targets. PNA probe showed no affinity towards non-complementary DNA. The non-specific binding of SNP containing DNA target was suppressed by the washing step of the beads by using sodium dodecylsulfate in blank buffer solution. Then, an electro-active intercalator, 7-dimethyl-amino-1,2-benzophenoxazinium salt (Meldola's blue, MDB) was introduced to the beads. MDB intercalated between the double-helix of the hybrid molecules on the beads. After removing the excessive MDB, the beads were collected from the solution by immersing a biotin modified carbon paste electrode into the solution. Specific hybridization between PNA probe and DNA target was determined by monitoring the voltammetric peak of MDB. Numerous factors affecting the MDB signal, such as target DNA concentration, intercalator concentration and accumulation time were investigated. MDB signal indicated a detection limit of 2 pM in connection with 20 min hybridization time. (C) 2004 Elsevier Ltd. All rights reserved.