The development of QCM-DNA sensor for p16 methylation detection in cholangiocarcinoma

Abstract

Promoter hypermethylation of tumor suppressor genes are associated with an epigenetically mediated gene silencing, which is a common feature in various human cancers including cholangiocarcinoma. Even though methylation-specific PCR (MSP) has been accepted for its sensitivity to detect methylation in cancerous tissues, this technique is time-consuming, labor-intensive and usage of carcinogenic chemicals. To overcome this limitation, DNA biosensor using quartz crystal microbalance (QCM) was developed in this study. QCM, a mass sensor, is rapidly becoming an important tool for biological analysis. p16 methylation in KKU-M213 CCA cell line was investigated as a model for protocol validation. In principle, specific biotinylated DNA probe were immobilized via avidin–biotin system on gold surface of a QCM device. The specific hybridization between immobilized probe with methylated and unmethylated p16 products were monitored by decreasing in frequency due to mass change. The results showed that the optimal concentrations of mercaptoproprionic acid (MPA), avidin, and 5’-biotinylated DNA probe were 10 mM, 1 mg/dL and 1 mM, respectively. The oscillation signal obtained from methylation or unmethylation positive samples were clearly discriminated from negative ones. The cut-off values were calculated from negative methylation and unmethylation at 30 Hz with preferable lower than 15 % precision. The QCM sensor can detect positive signal at 1,000 fold dilution compared to agarore gel electrophoresis. No cross-hybridization was observed when unmethylation product was performed on methylation sensor and vice versa. Moreover, QCM can be reused for at least 3 times with considerable signal reduction. In conclusion, our developed QCM sensor for p16 methylation illustrated an acceptable performance compared to conventional method. The detection process can be reduced within 5 minutes. However, this prototype sensor as well as mini-PCR integration device for real time analysis should be further developed.