Analytical Sciences

Direct Detection of Single Nucleotide Polymorphism (SNP) with Genomic DNA by the Ferrocenylnaphthalene Diimide-based Electrochemical Hybridization Assay (FND-EHA)

Takahiko NOJIMA,*¹ Kenichi YAMASHITA,*¹ Atsuko TAKAGI,*² Makoto TAKAGI,*¹ Yasuyuki IKEDA,*³ Hiroki KONDO,*⁴ and Shigeori TAKENAKA*⁴ 

*1 Department of Applied Chemistry, School of Engineering, Kyushu University, Fukuoka 812-8581, Japan
*2 Department of Etiology and Pathophysiology, National Cardiovascular Center Research Institute, Fujishirodai, Suita, Osaka 565-8565, Japan
*3 Department of Pharmacology, National Cardiovascular Center Research Institute, Fujishirodai, Suita, Osaka 565-8565, Japan
*4 Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Iizuka 820-8502, Japan

A ferrocenylnaphthalene diimide-based electrochemical hybridization assay (FND-EHA) was applied to the direct detection of a C-to-G transition in a codon (TCA) for Ser-447 of the human lipoprotein lipase (LPL) gene, which resulted in the termination of the LPL protein there. Either one of two 13-meric oligonucleotide probes, S447 WT and S447X MT, representing sequences complementary to those of the wild type (WT) and mutated (MT) forms, was immobilized on a gold electrode, followed by hybridization with chromosomal DNA extracted from human leukocytes under the condition in which both WT- and MT-type sequences can form a duplex. These two electrodes were soaked in an electrolyte containing FND under a condition [0.1 M HOAc/KOAc (pH 5.6) containing 0.1 KCl and 0.05 mM FND at 40°C], in which only the MT duplex could undergo dissociation. FND was concentrated in proportion to the amount of the duplex formed on the electrode to give rise to a current signal. The electrochemical signal ratios obtained for WT/WT, WT/MT and MT/MT were close to the theoretical 2:1:0 with the S447 WT-modified electrode, and was again close to 0:1:2 with the S447X MT-modified one.

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