A Site-specific DNA Methylation Biosensor For The Determination Of MiR-34a Promotor Based On Lateral Flow Assay

Aberration in DNA methylation,particularly in the promoter of cancer-prognostic genes,is a significant marker in tumorigenesis and has been served as a predictive biomarker for cancer diagnosis and prognosis.Although numerous DNA methylation identification techniques have been developed such as methylation sensitive high-resolution melting curves and bisulfite sequencing,sophisticated instruments are required as well as the cumbersome operations,which severely hinder their application in basic medical institution.Therefore,there is an urgent requirement to develop a portable,rapid and sensitive strategy for accurate methylation detection with specific cancer-prognostic gene,which caters to the popularization of precision medicine.In this study,a site-specific biosensor for DNA methylation determination has been established based on lateral flow assay.In terms of design strategy,the amplification-refractory mutation system was introduced into the design of methylation-specific primers.The pattern of target Cp G site can be detected through a pair of PCR amplification reactions.By introducing bio-labeled oligonucleotides into PCR,the amplicons can be recognized and captured by gold magnetic nanoparticle(GMNP)for the interpretation of results in this biosensor.Working as a signal probe,the optical property of GMNP allows the amplicons to be interpreted with naked eyes in 5 min avoiding any complex equipment and cumbersome operation after PCR.Moreover,by virtue of the magnetic property of GMNP,the detection result can be explained by a magnetometer for digital interpretation automatically.The introduction of tailor-made primer sets makes 0.1%methylated variants can be accurately distinguished in the presence of a large excess of unmethylated variants as strong interferential background and vice versa at target cytosineguanine dinucleotide.After bisulfite modification,a distinct signal can be observed with as low as 0.01 pg variants for both visual and magnetic analysis within 90 min.As a significant tumor suppressor gene,the promoter methylation status of miR-34 a is accurately determined with not only cell lines but also clinical samples,which demonstrates the great potential of this biosensor in cancer diagnosis and prognosis.