Abstract
Telomerase is an enzyme that elongates repeated six bases of TTAGGG of the telomere DNA, and is known to be expressed in tumor cells, but not in normal cells. For this reason, this enzyme is expected to serve as a new tumor marker. The telomerase activity has been detected by the telomerase repeat amplification protocol (TRAP), which involves a tedious polymerase chain reaction (PCR) and gel electrophoresis. We have been developing an electrochemical telomerase assay with ferrocenylnaphthalene diimide (FND) as an electrochemical indicator. Where telomerase activity is present in the sample, the telomerase substrate (TS)-primer immobilized on the electrode is elongated to form a telomeric repeat sequence ; the resulting products form a tetraplex DNA. FND binds to the tetraplex DNA thus formed to give rise to an electrochemical signal whose magnitude reflects the telomerase activity. We tested saliva, oral epithelial cells, and solid tumor for telomerase activity for diagnosis of tongue cancer using disposable electrode chips coupled with a FND derivative. The samples were lysed, and supernatants (2000 and 200 ng of protein) were used in TRAP and the FND-based electrochemical assay with Osteryoung square wave voltammetry (SWV). In both assays, all of the samples from a solid tumor turned out to be telomerase-positive. However, the telomerase activity was detected at a higher rate, even in saliva and oral epithelial cells in the latter assay than in the former. In conclusion, the FND-based electrochemical telomerase assay was more sensitive than TRAP in terms of the detection of cancerous samples taken from tongue cancer patients. This electrochemical method will enable early diagnosis of tongue cancer in clinical dentistry.
