Publication 682

Anal. Chem., 83 (5), 1815-1821, 2011 DOI:10.1021/ac1033374
Real-time electrochemical PCR with a DNA intercalating redox probe

Laboratoire d’Electrochimie Moléculaire, UMR CNRS 7591, Université Paris Diderot, 15 rue Jean-Antoine de Baïf, 75205 Paris Cedex 13, France

The proof-of-principle of a nonoptical real-time PCR method based on the electrochemical monitoring of a DNA intercalating redox probe that becomes considerably less easily electrochemically detectable once intercalated to the amplified double-stranded DNA is demonstrated. This has been made possible thanks to the finding of a redox intercalator that (i) strongly and specifically binds to the amplified double-stranded DNA, (ii) does not significantly inhibit PCR, (iii) is chemically stable under PCR cycling, and (iv) is sensitively detected by square wave voltammetry during PCR cycling. Among the different DNA intercalating redox probes that we have investigated, namely, methylene blue, Os[(bpy)₂phen]²⁺, Os[(bpy)₂DPPZ]²⁺, Os[(4,4′-dimethyl-bpy)₂DPPZ]²⁺ and Os[(4,4′-diamino-bpy)₂DPPZ]²⁺ (with bpy = 2,2′-bipyridine, phen = phenanthroline, and DPPZ = dipyrido[3,2-a:2′,3′-c]phenazine), the one and only compound with which it has been possible to demonstrate the proof-of-concept is the Os[(bpy)₂DPPZ]²⁺. In terms of analytical performances, the methodology described here compares well with optical-based real-time PCRs, offering finally the same advantages than the popular and routinely used SYBR Green-based real-time fluorescent PCR, but with the additional incomes of being potentially much cheaper and easier to integrate in a hand-held miniaturized device.