M.J. Heller, S. Ibsen, J. Wright, J. Lewis
University of California San Diego,
Keywords: DEP, exosomes, DNA, RNA, drug delivery nanoparticles, cancer, diagnostics
Summary:The rapid isolation and detection of circulating cell free (ccf) DNA, ccf-RNA, exosome biomarkers and drug delivery nanoparticles is critically important for enabling new “liquid biopsy” cancer diagnostics and patient therapy monitoring. We have now demonstrated a novel dielectrophoretic (DEP) microarray based approach that allows ccf-DNA, ccf-RNA, exosomes as well as drug delivery nanoparticles to be isolated in about 10-20 minutes directly from small volumes (25-100ul) of blood, plasma or serum. Ccf-DNA isolated from chronic lymphocytic leukemia (CLL) patient blood samples and plasma samples by DEP were PCR amplified to identify the VHL genotype and then sequenced. The DNA sequencing results for ccf-DNA isolated by DEP were compared to two gold standard methods for CLL analysis. In further biomarker related work, the DEP microarray devices were used to carry out the rapid isolation of glioblastoma exosomes from 50 µL of human plasma. When an AC field is applied, the exosomes (100nm to 150nm size lipid/protein/RNA vesicles) preferentially concentrated into the DEP high-field region around the microelectrodes in 15-20 minutes. A buffer wash was then used to remove the bulk plasma materials from the device, while the exosomes remained concentrated on the microelectrodes. RT-PCR analysis of the ccf-RNA from exosomes showed presence of both mRNA (-actin) and mRNA for the cancer-specific EGFRvIII. Finally, the DEP technology was used to carry out the recovery and analysis of drug delivery nanoparticles from undiluted human plasma samples. The nanoparticles are recovered from plasma through a dielectrophoresis separation force that is created by innate differences in the dielectric properties between the unaltered nanoparticles and the surrounding plasma. The DEP was successful in isolating a wide range of drug delivery nanoparticles of different morphologies and materials, including low density nano-liposomes. Thus, the new DEP technology and devices set the stage for “seamless sample to answer” molecular diagnostic and therapy monitoring systems which will allow a variety of important cancer and other disease biomarkers, as well as drug delivery nanoparticles, to be rapidly isolated and analyzed directly from whole blood and other clinical samples.