Detection of a K-ras point mutation employing peptide nucleic acid at the surface of a SPR biosensor

A fundamental study of the usage of peptide nucleic acid (PNA) and DNA at the surface of a surface plasmon resonance (SPR) biosensor for detecting a specific point mutation in the K-ras gene for early detection of cancers is reported. The extents of hybridization using DNA analytes of different length which are targets were studied at 25 and 40°C. The 15mer PNA ligand immobilized to the surface of the sensor had a high ability to detect a 11mer DNA analyte point mutation at 25 and 40°C. On the contrary, when DNA was used as a ligand, detection was only confirmed at 40°C which indicates that the difference of melting temperature (T_m) between the complementary and mismatched analyte was a key factor for point mutation detection. This also implied the decrease of T_m by immobilizing a probe to a surface since it was below the one in solution. Therefore, effects from the surface are seen in this system and the surrounding environment makes a difference to the discrimination process. To understand the process in detail, kinetic analysis was performed and association constants were found to be larger for DNA ligands implying the effect coming from the sensor surface. In this case negative charges of the carboxyl groups of the matrix had a tendency to repel negatively charged DNA ligands. Affinity constants (K_A) were calculated and as expected, the ratio of K_A between the complementary and mismatched analyte was of the order of 10² for PNA interacting with 11mer analytes, making it a promising probe for genetic diagnosis. These results indicate the importance of the effect of analyte length, temperature and the environment surrounding the ligand for accomplishing an all or none single base mismatched detection system.