Ferrocenylnaphthalene Diimide-Based Electrochemical Hybridization Assay for a Heterozygous Deficiency of the Lipoprotein Lipase Gene

An electrochemical hybridization assay has been devised that enables the rapid analysis of a heterozygous deficiency of the human lipoprotein lipase (LPL) gene. PCR products of 350 base pairs (bp) containing the wild-type sequence, a mutated G818 → A transition or a G916 deletion of the LPL gene were subjected to hybridization with a probe DNA of 13 or 15 bases that represented either the wild-type or the mutated sequence immobilized on a gold electrode. The differential pulse voltammetry of the electrode before and after hybridization was determined in the presence of ferrocenylnaphthalene diimide (FND) at 460 mV. The measured change in peak current, Δi, was defined by (i − i o)/i o × 100%, where i o and i represent the current before and after hybridization, respectively. Matched combinations of sample and probe gave Δi values of 40−90%, whereas mismatched combinations gave values of 20−35%, enabling the discrimination of matched hybrids from mismatched ones across a slim margin. Because the heterozygote contains both the wild-type and mutated sequences, however, it alone gives large Δi values with both the wild- and mutant-type probes. This system was validated on 10 unknown samples of each of the two types of LPL mutation, which were correctly identified in every case.