DNA-redox conjugate for applications to electrochemical gene sensing

Abstract

Two examples of an electrochemical gene-sensing system are reported. They are built based on the chemistry of a DNA probe, a DNA-modified electrode and, especially, DNA conjugate formation through a DNA-DNA and a DNA-small molecule interactions to assure electrochemcial sensing. A 16-mer oligodeoxynucleotide (ODN) having five successive phophorothioate units on its 5'-terminus (s16) was prepared and covalently immobilized onto gold electrode surfaces through chemisorption. A ferrocenyl ODN (f12) was synthesized by a coupling reaction between amino-terminated ODN with an activated ester of ferrocenecarboxylic acid. The f12 was subsequently annealed with its complementary strand, p19, which contains the complemental sequence to s16. The resulting double-strand, p19-f12, was used for hybridization experiments. A treatment of the s16-modified electrode turned it to be electroactive due to s16-p19-f12 sandwich-type complex formation. Cyclic voltammetric (CV) measurements in an aqueous KCl solution showed reversible redox waves due to the redox reaction of the ferrocenyl moiety. On the other hand, a s16-modified electrode treated with the mismatch control, m19-f12, also showed redox waves, but only slightly (ca. 5% to p19-f12). These results indicate that the present sensing system is fundamentally applicable to the electrochemcial detection of specific genes. Next, a ferrocenyl derivative of psoralen (FcPso) is described. Psoralen is a class of intercalator which forms a photoadduct with the pyrimidine bases upon UV radiation; FcPso is expected to be useful for the electrochemical tagging of DNA. FcPso was synthesized by a reaction of 4'-chloromethylated 4,5',8-trimethylpsoralen with N,N-dimethylaminomethylferrocene. A 5'-terminally thiolated 12- mer ODN (e12) was immobilized on a gold electrode surface through chemisorption. The e12-electrode was annealed with the complementary strand, t12. Although CVs measured in an aqueous KCl solution only gave capacitive currents, a FcPso treatment turned the electrode system redox actively; the CV peak currents due to the Faradaic reaction of the ferrocene moiety showed a linear dependence on the sweep rate of the electrode potential, indicating a surface process. On the other hand, almost no faradaic response was associated for FcPso treatment of the e12-electrode. The potential application of the electrochemcial detection of a specific gene was successfully demonstrated. As a basis of gene-sensor applications, a detailed characterization of the immobilization chemistry is also presented. The results concerning IR spectral measurements and a microgravimetric analysis using a quartz-crystal microbalance are described.