(185f) AC Electrokinetic Isolation and Detection of Cell Free DNA, RNA and Exosome Biomarkers for Sample to Answer Molecular Diagnostics

Circulating cell free (ccf) DNA, RNA and exosomes are now important biomarkers for liquid biopsy cancer diagnostics, and also hold great promise for early cancer detection. Nevertheless, the isolation of these biomarkers from patient samples requires relatively complex, time consuming and expensive procedures which greatly limits their practical use for most cancer diagnostic applications. New AC dielectrophoretic (DEP) microarray/chip devices now allow 15-20-minute isolation of cancer related ccf-DNA, RNA and exosome biomarkers from 20-50ul of blood, plasma or serum. After isolation of the biomarkers, specific fluorescent dyes can be used first to simultaneously detect the different biomarker levels directly on the chip (in-situ). In a subsequent step, immunofluorescent analysis can be carried out to identify specific protein biomarkers on the exosomes. Finally, the ccf-DNA and RNA (mRNAs and miRNAs release from the exosomes) can be eluted from the DEP chip, and PCR and sequencing analysis carried out to identify the cancer-related point mutations and other polymorphisms, as well as to further verify the tissue origin of the biomarkers. In the case of our Chronic Lymphocytic Leukemia studies, final PCR and DNA sequencing results for CLL Ig heavy-chain variable regions in the IGHV genes and SNPS isolated by DEP were found to be exactly comparable to two much more complex and time consuming â??gold standardâ? procedures. In the case of glioblastoma exosomes isolated from plasma, exosome-specific surface and interior proteins CD63 and TSG101 could be detected by immunofluorescence, and mutated EGFRvlll mRNA was detected by RT-PCR. Finally, the exosomal related protein biomarker Glypican-1 could be isolated from pancreatic cancer patient plasma samples by DEP and detected on-chip by immunofluorescence. Thus, DEP represents a powerful new minimally invasive technology for cancer diagnostics that is particularly well suited for the rapid isolation of cell free nucleic acid and exosome biomarkers. The technology is setting the stage for seamless sample to answer liquid biopsy, cancer patient therapy monitoring and ultimately for early disease detection.