Dip

Disease

  • The disease to be diagnosed is a cancer that has metastasized and is travelling in the bloodstream. Specifically, this cancer is a type of cancer that has the potentially to grow into tumors around the body. No specific cancer has been assigned yet, but it must follow these parameters.

 

Analyte

  • The analyte to be isolated and examined is DNA. Cancer cells’ DNA is typically marked by hypermethylation of tumor suppressor genes such as the p53 protein coding gene TP53, and the hypomethylation of oncogenes such as those in the RAS subfamily. Methylation of a CpG group in DNA typically marks suppression of a gene, so in this case both actions lead to cancerous cells.

 

Sample/Reagent Loading and Transport

  • The blood sample and PCR agents will be loaded into the nanolyser via a set of inlet ports located on one side of the chip. Transportation will occur using a motor protein (kinesin) driven unidirectional transport system. The array of kinesin proteins will be aligned using a flow field. See nannotated bibliography for reference.

 

DNA Isolation

  • Cells will be lysed and DNA will be captured with the use of poly (N-isopropylacrylamide) brushes tethered to a silicon surface. The brushes change polarity after a certain temperature (~40 deg C) to allow for capture and release of DNA. See nannotated bibliography for reference.

 

Results and Analysis

  • An Hpall tiny fragment Enrichment by Ligation Mediated PCR Assay (HELP Assay) will be conducted on the collected DNA to determine the relative amounts and location of methylation. Hpall digests 5’-CCGG-3’ segments that are not methylated, thus allowing for a determination of methylated versus unmethylated CpG sites and its frequency. Recent publications showed a computerized analyzer of HELP Assay data written in the R programming language.

Summarized Algorithm

  1. Blood sample and HELP Assay reagents are loaded via inlet ports on side of nanolyser.
  2. Motor protein based transport induced by flow field transports blood sample to cell lysing mechanism.
  3. Lysed cells are sent through DNA isolator.
  4. Temperature is raised to release DNA from isolator.
  5. DNA and reagents are transported to HELP Assay area and mixed with reagents.
  6. HELP Assay is ran and results are analyzed using computer.

IMG_1403 IMG_1402