Thesis (M.Sc.)--Chulalongkorn University, 2012

In this work, a novel method for detection of high-risk human papillomavirus (HPV) DNA type 16 employing pyrrolidinyl polyamide nucleic acid (acpcPNA) probe coupled with electrochemical detection was developed. A capture probe was designed to bind specifically to HPV DNA type 16. The change of electrochemical signal of redox-active acpcPNA probe was observed. Anthraquinone was used as a redox-active label, which was attached at the N-terminus of PNA probe. The AQ-modified acpcPNA (PNA-AQ) was characterized by MALDI-TOF MS and thermal denaturation study with a complementary synthetic DNA target corresponding to the HPV DNA sequence (Tm = 69.9 oC). Chitosan-modified screen-printed carbon electrode was used in this study. The PNA-AQ probe was covalently immobilized onto the electrode surface using glutaraldehyde as a cross linking agent. Electrochemical detection was performed under optimal parameters using a square-wave voltammetric method. As a result, the probe signal decreased when exposed to the target DNA. The developed method showed a linear range between 0.02 –12 µM of DNA target with a correlation coefficient of 0.996. The limit of detection (LOD) and limit of quantitation (LOQ) was found to be 0.004 µM and 0.014 µM, respectively. Finally, this novel method was successfully applied to detect the HPV DNA type 16 in cell-lines sample (SiHa). The concentration of HPV DNA that this method can detect was found to be 1.25 ± 0.08 µM. This developed method produced results in good agreement with the standard marker. Moreover, the sensor probe has very high selectivity over non-complementary oligonucleotides, including HPV type 18, 31 and 33 DNA. This detection method can serve as a novel and inexpensive tool for early stage detection of cervical cancer, which is in demand in developing countries.