METHYL-SENTRY: Proposed feasibility study of a nanopore diagnostic tool to detect hypermethylated biomarkers in cell free DNA indicative of cancer state Early detection of cancer is correlated with improved outcomes and reduced morbidity due to more timely treatment. Ideally, detection strategies should be non-invasive, rapid, and easy to deploy in a clinical setting. Circulating cell-free DNA (cfDNA) is released by normal cells, however, in cancer patients a portion of cfDNA comes from tumor cells (circulating-tumor DNA or ctDNA). As tumor cells divide faster than normal cells, cancer patients typically have a high level of cfDNA in serum because of necrosis or apoptosis, with a greater proportion of cfDNA attributable to tumors. Detection of the ctDNA fraction of cfDNA presents an excellent opportunity for a non-specific, blood- or urine-based screening, referred to as a “liquid biopsy”. One of the most readily detected changes in the DNA of cancer patients is alterations in the pattern of genome methylation, a significant control mechanism of gene expression. However, there is limited diagnostic information from detecting an increase in cfDNA, and later approaches targeted ctDNA and cancer-specific alterations to this DNA. Here we propose the use of solid-state nanopores for detection of hypermethylated cytosine adjacent guanine (CpG) dinucleotide clustered islands (CGI) in cfDNA as a sensitive and non-specific technique for detection of cancer from blood or urine samples. As with protein nanopores, solid-state nanopores allow single molecule measurements with translocation of the DNA strand through the pore resulting in a unique electrical signal. Sequences will be evaluated in the presence of methyl binding domain (MBD) protein for selectively binding to methylated CpG domains and further amplification of nanopore derived quantification signal. In our proposed method, the electrical signature is representative of the entire molecule, instead of a single nucleotide, for detection of hypermethylated CGIs as a cancer biomarker. The proposed work is supported by three Technical Objectives (TOs). TO1 is to determine the sensitivity of the method for the number of methylated CpG per CGI fragment. TO2 will determine the ability of our method to detect an increase in hypermethylation of CGIs above the background of partially methylated cfDNA in a control sample. TO3 will determine the feasibility and sensitivity of this system in a series of clinical cancer mimic samples. In contrast to previously developed conventional methods, our proposed method would be fast, sensitive, and independent of the underlying DNA sequence so that it could be used for cancer detection regardless of mutations in the target sequence. This solid-state based method can ultimately be commercialized into a simple to use clinical setting general cancer detection and treatment progression diagnostic.