Rajaram Krishnan Benjamin D. Sullivan Robert L. Mifflin Sadik C. Esener Michael J. Heller University of California, San Diego, Department of Bioengineering/Department of Electrical and Computer Engineering, La Jolla, CA, USA Received January 14, 2008 Revised February 27, 2008 Accepted February 28, 2008 Alternating current electrokinetic separation and detection of DNA nanoparticles in high-conductance solutions In biomedical research and diagnostics, it is a significant challenge to directly isolate and identify rare cells and potential biomarkers in blood, plasma and other clinical samples. Additionally, the advent of bionanotechnology is leading to numerous drug delivery approaches that involve encapsulation of drugs and imaging agents within nanoparticles, which now will also have to be identified and separated from blood and plasma. Alternating current (AC) electrokinetic techniques such as dielectrophoresis (DEP) offer a particularly attractive mechanism for the separation of cells and nanoparticles. Unfortunately, present DEP techniques require the dilution of blood/plasma, thus making the technology less suitable for clinical sample preparation. Using array devices with microelectrodes over- coated with porous hydrogel layers, AC electric field conditions have been found which allow the separation of DNA nanoparticles to be achieved under high-conductance (ionic strength) conditions. At AC frequencies in the 3000 Hz to 10 000 Hz range and 10 volts peak-to-peak, the separation of 10-mm polystyrene particles into low field regions, and 60-nm DNA-derivatized nanoparticles and 200-nm nanoparticles into high-field regions was carried out in 149 mM 1 6PBS buffer (1.68 S/m). These results may allow AC electro- kinetic systems to be developed that can be used with clinically relevant samples under physiological conditions. Keywords: Alternating current electrokinetics / Dielectrophoresis / DNA / High-conductance solution / Nanoparticles DOI 10.1002/elps.200800037 Electrophoresis 2008, 29, 1765–1774 1765 1 Introduction In clinical diagnostics and many areas of biomedical re- search it is both important and frequently a challenge to separate and identify rare cells (cancer), low numbers of bacteria and virus, low concentrations of DNA biomarkers, antibodies and other entities in complex samples like blood [1], plasma [2], serum [3], saliva [4] and urine [5]. Additionally, the advent of bionanotechnology is leading to numerous drug delivery approaches that involve encapsulation of drugs and imaging agents within nanovesicles and nanoparticles [6]. Thus, it will now also be important to identify and moni- tor residual nanovesicles and nanoparticles that remain in the blood. A variety of physical, electronic and biological methods and techniques can be used for the isolation of cells, bio- markers and nanoparticles from complex samples like blood. These include centrifugation, gel filtration, affinity binding, magnetic beads, electrophoresis, flow cytometry and various combinations thereof incorporated into lab-on-a-chip, microfluidic devices and sample to answer systems [7, 8]. Nevertheless, many of these techniques remain relatively time-consuming processes that are not without problems and limitations. Alternating current electrokinetic tech- niques which involve the use of alternating current (AC) fields to manipulate particles offer a particularly attractive mechanism for rapid separation and analysis of cells [9–11], biomarkers such as cell-free circulating high-molecular weight DNA [12–15] and proteins [16], and ultimately drug Correspondence: Professor Michael J. Heller, UCSD Department of Bioengineering, PFBH Rm 429, 9500 Gilman Drive, La Jolla, CA, 92093-0412, USA E-mail: mheller@bioeng.ucsd.edu Fax: 11-858-822-5689 Abbreviations: AC, alternating current; DEP , dielectrophoresis; pk-pk, peak to peak Fast Track  2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com