UC San Diego

Cell-free circulating (CFC) DNA is now considered an important biomarker for early detection of cancer, residual disease, monitoring chemotherapy and other aspects of cancer management. The isolation of CFC-DNA from plasma as a "liquid biopsy" may begin replacing more invasive tissue biopsies as a means to detect and analyze cancer mutations. Unfortunately, conventional techniques for the isolation of CFC-DNA from plasma require a relatively time consuming and complex process which would rule out their use for point of care diagnostic applications. This work demonstrated the rapid isolation and detection of both single-stranded and double-stranded HMW-DNA and 40 nm nanoparticles directly from whole blood and buffy coat blood samples. We then went on to show the rapid isolation and detection of SYBR Green stained CFC- DNA from 20 [mu]L whole blood samples from Chronic Lymphocytic Leukemia (CLL) patients, as well as isolation of cell-free mitochondria and virus from high conductivity buffers. To investigate the potentially clinical utility of this technology, we isolated CFC-DNA from fresh blood samples of fifteen CLL patients and three healthy individuals. CFC-DNA from 25 [mu]L (a drop) of blood was separated and concentrated into DEP high-field regions in about three minutes and held while blood cells, proteins and other biomolecules were removed by a fluidic wash. Concentrated CFC-DNA was detected by fluorescence and then eluted for PCR and DNA sequencing. The complete process, blood to CFC-DNA for PCR, spanned less than 10 minutes. Eluted CFC-DNA, from 5 [mu]l of the original CLL blood sample, was amplified by PCR using IGVH-specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone. The PCR results obtained by DEP from CLL blood were comparable to results obtained using conventional sample preparation of CFC-DNA starting with one ml of plasma and the sequencing results were accurate for all 15 patient samples. The ability of DEP to provide rapid isolation of CFC-DNA from the equivalent of a drop of blood represents a major step forward in the mission to create viable point of care cancer diagnostics and patient monitoring. This technology is applicable to a wide variety of biomarkers in biological and environmental samples and has broad implications as low-complexity and easily integrated front-end sample processing solution