Real-Time Electrochemical PCR with a DNA Intercalating Redox Probe

Abstract

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)(2)phen](2+), Os[(bpy)(2)DPPZ](2+), Os[(4,4'-dimethyl-bpy)(2)DPPZ](2+) and Os[(4,4'-diamino-bpy)(2)DPPZ](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)(2)DPPZ](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.

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“…In some cases, although the molecules give good electrochemical signals, their affinity towards DNA is low. The three most effective and widely used electroactive DNA intercalating molecules for detection of DNA amplification are methylene blue [45,46,47], osmium bipyridine-based complex and bisbenzimidazole trihydrochloride (Hoechst 33258) [48,49,50]. Since this electrochemical technique is simple, it is considered a suitable candidate for development of an on-site gene detection device.…”

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“…Therefore, an increase in the peak current is proportional to the increase in the concentration of DNA double helix. Hoechst 33258 is selected as an electroactive indicator to avoid tedious procedures in order to label dsDNA [29]. Fig.…”

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“…Recently, electrochemical real time dsDNA detection has been reported using electrochemically active intercalator derivatives. [23][24][25] The dsDNA was detected by the decrease of the diffusion coefficient of redox intercalator upon binding to dsDNA, resulting in the current decrease. This system is regarded as a homogenous electrochemical dsDNA assay, but it is "signal off"-type detection.…”