Quantitative analysis for solid-phase hybridization reaction and binding reaction of DNA binder to hybrids using a quartz crystal microbalance

A 20-mer DNA probe complementary to a part of an oncogene v-myc, having a mercaptohexyl group at the 5′-end, was immobilized on a gold electrode of a quartz crystal microbalance (QCM). The quantity of the probe immobilized on the electrode depended on the pH. Under acidic or basic immobilization conditions, the probe was immobilized more than under neutral conditions. The largest quantity of the target DNA containing v-myc was hybridized when the probe was immobilized under neutral conditions. Using the QCM on which the probe was immobilized under neutral conditions, the quantity of the hybridized target increased with the quantity of the immobilized probe. In the region where the QCM was saturated with immobilized probes, around 70% of the given target was hybridized, and the ratio of target/probe was about 1:100. After the hybridization, the QCMs were reacted with Hoechst 33258 (a DNA binder), which bound to the DNA hybrids formed on the QCMs. The quantity of the bound Hoechst 33258 increased with the quantity of the hybridized target, and the anodic current derived from linear sweep voltammetry of the bound Hoechst 33258 also increased with the quantity of the hybridized target. These results suggested that QCMs could be applied to quantitative analysis for the solid-phase hybridization and the binding of a DNA binder to hybrids.