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Circulating cell-free DNA (ccf-DNA) is becoming an important biomarker for cancer diagnostics and therapy monitoring. The isolation of ccf-DNA from plasma as a \"liquid biopsy\" may begin to replace more invasive tissue biopsies for the detection and analysis of cancer-related mutations. Conventional methods for the isolation of ccf-DNA from plasma are costly, time-consuming, and complex, preventing the use of ccf-DNA biomarkers for point-of-care diagnostics and limiting other biomedical research applications. We used an AC electrokinetic device to rapidly isolate ccf-DNA from 25 μL unprocessed blood. ccf-DNA from 15 chronic lymphocytic leukemia (CLL) patients and 3 healthy individuals was separated into dielectrophoretic (DEP) high-field regions, after which other blood components were removed by a fluidic wash. Concentrated ccf-DNA was detected by fluorescence and eluted for quantification, PCR, and DNA sequencing. The complete process, blood to PCR, required <10 min. ccf-DNA was amplified by PCR with immunoglobulin heavy chain variable region (IGHV)-specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone, and then sequenced. PCR and DNA sequencing results obtained by DEP from 25 μL CLL blood matched results obtained by use of conventional methods for ccf-DNA isolation from 1 mL plasma and for genomic DNA isolation from CLL patient leukemic B cells isolated from 15-20 mL blood. Rapid isolation of ccf-DNA directly from a drop of blood will advance disease-related biomarker research, accelerate the transition from tissue to liquid biopsies, and enable point-of-care diagnostic systems for patient monitoring.

Circulating cell-free (ccf) DNA has become an important biomarker for the early detection, monitoring, and treatment of cancers. However, the current gold standard methods of isolating ccf-DNA from blood or plasma are labor intensive and time consuming. The complex sample processing procedures may cause degradation or loss of ccf-DNA, and thus greatly inhibit the use of ccf-DNA as a target biomarker for point of care (POC) diagnostics. Therefore, it is essential to develop a rapid and inexpensive ccf-DNA extraction method in order to use ccf-DNA as a biomarker for clinical applications. The work described in this thesis demonstrates a dielectrophoretic- (DEP) based method for the rapid isolation of ccf-DNA from undiluted whole blood and plasma samples collected from chronic lymphocytic leukemia (CLL) patients. It is demonstrated that DEP can recover ccf-DNA from 25 µL of blood and plasma in less than 15 minutes. To investigate the potential of DEP for use in clinical applications, the dielectrophoreticly recovered ccf-DNA from blood and plasma of CLL patients was amplified using polymerase chain reaction (PCR) and sequenced for CLL specific mutations. The results of the genetic analysis were found to be comparable to sequencing results obtained from ccf-DNA isolated by conventional golden standard methods as well as the DNA extracted directly from B-cells. The ability of DEP-based technology to rapidly isolate ccf-DNA from small volumes of unprocessed blood shows the potential to accelerate sample processing and enable the use of ccf-DNA as a specific biomarker target for point of care diagnostics.