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Background: 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., Methods: 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., Results: 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., Conclusions: 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.

Selection and adjustment of proper physical parameters enables rapid DNA transport, site selective concentration, and accelerated hybridization reactions to be carried out on active microelectronic arrays. These physical parameters include DC current, voltage, solution conductivity and buffer species. Generally, at any given current and voltage level, the transport or mobility of DNA is inversely proportional to electrolyte or buffer conductivity. However, only a subset of buffer species produce both rapid transport, site specific concentration and accelerated hybridization. These buffers include zwitterionic and low conductivity species such as: d- and l-histidine; 1- and 3-methylhistidines; carnosine; imidazole; pyridine; and collidine. In contrast, buffers such as glycine, beta-alanine and gamma-amino-butyric acid (GABA) produce rapid transport and site selective concentration but do not facilitate hybridization. Our results suggest that the ability of these buffers (histidine, etc.) to facilitate hybridization appears linked to their ability to provide electric field concentration of DNA; to buffer acidic conditions present at the anode; and in this process acquire a net positive charge which then shields or diminishes repulsion between the DNA strands, thus promoting hybridization.

The morbidity and mortality rates of cytomegalovirus (CMV) infections, including pneumonitis in bone marrow transplant patients, are well documented and yet no rapid, sensitive diagnostic tool is available. To test the polymerase chain reaction as such a diagnostic tool, formalin-fixed, paraffin-embedded open lung biopsy material was selected from post-bone marrow transplant patients in whom pulmonary parenchymal changes were evident but viral inclusions were not seen. As a control, other immunosuppressed (non-bone marrow transplant) patients and low-risk nonimmunosuppressed patients were studied in a similar manner. Viral culture results and clinical data were available on all the high-risk patients. Two of 15 high-risk patients were found to have amplifiable CMV DNA despite the lack of histologic viral inclusions. One of these patients had been treated recently for CMV pneumonia, and the other had a positive culture of the lung specimens for CMV 13 days after biopsy. None of 12 control patients had amplifiable CMV DNA. These data indicate that the polymerase chain reaction is more sensitive than histologic examination and at least as sensitive as viral culture without evidence of false-positive results.