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Abstract
The DNA‐modified membrane electrode was prepared by casting a mixture of nitrocellulose (NC) with target DNA (tDNA) in organic solvent on glassy carbon electrode (GCE). Unlabeled polymerase chain reaction (PCR)‐amplified human genomic sequence (628 bp) or synthetic oligodeoxynucleotides (ODNs) were used as tDNAs, creating a recognition layer. Biotinylated ODNs were used as hybridization probes to recognize specific nucleotide sequences. The hybridization events were detected via an enzyme‐linked electrochemical assay involving binding of streptavidin‐coupled alkaline phosphatase (SALP) to the biotin labels of the probe bound to tDNA. After the probe hybridization and SALP binding, the electrode was immersed into an electroinactive enzyme substrate (1‐naphthyl phosphate). The alkaline phosphatase converted the inactive substrate into electroactive 1‐naphthol that penetrated through the NC membrane to the GCE surface and was subsequently detected using an anodic voltammetric signal. The optimized method offered a good discrimination between complementary and nonspecific DNAs and yielded well‐defined responses for both single‐copy and repetitive tDNA sequences. In contrast to previously published methods using electrodes with mechanically attached membranes, the previously mentioned electrode is easily amenable to parallel DNA analysis.
On the occasion of the eightieth birthday of Professor Petr Zuman.
Acknowledgments
This work was supported by a purpose‐endowment from the Ministry of Industry and Trade of the Czech Republic (project 1H‐PK/42), by grants No.03‐03‐33116 from the Russian Foundation of Fundamental Researches, Nos. A4004402 and S5004355 from the Grant Agency of the Academy of Sciences of the Czech Republic, and by grant No. AVOZ50040507. The authors thank Dr. Z. Pechan for his assistance in the initial stage of this work, to Dr. P. Pecinka for preparation of the fra PCR products, and to Synthesia, Pardubice, CR and Wolff Cellulosics, Walsrode, Germany, for the donation of samples of nitrocellulose.
Notes
On the occasion of the eightieth birthday of Professor Petr Zuman.