Improved Bacterial and Viral Recoveries from 'Complex' Samples using Electrophoretically Assisted Acoustic Focusing (2008)

Automated front-end sample preparation technologies can significantly enhance the sensitivity and reliability of biodetection assays [1]. We are developing advanced sample preparation technologies for biowarfare detection and medical point-of-care diagnostics using microfluidic systems with continuous sample processing capabilities. Here we report an electrophoretically assisted acoustic focusing technique to rapidly extract and enrich viral and bacterial loads from 'complex samples', applied in this case to human nasopharyngeal samples as well as simplified surrogates. The acoustic forces capture and remove large particles (&gt; 2 {micro}m) such as host cells, debris, dust, and pollen from the sample. We simultaneously apply an electric field transverse to the flow direction to transport small ({le} 2 {micro}m), negatively-charged analytes into a separate purified recovery fluid using a modified H-filter configuration [Micronics US Patent 5,716,852]. Hunter and O'Brien combined transverse electrophoresis and acoustic focusing to measure the surface charge on large particles, [2] but to our knowledge, our work is the first demonstration combining these two techniques in a continuous flow device. Marina et al. demonstrated superimposed dielectrophoresis (DEP) and acoustic focusing for enhanced separations [3], but these devices have limited throughput due to the rapid decay of DEP forces. Both acoustic standing waves and electric fields exert significant forces over the entire fluid volume in microchannels, thus allowing channels with larger dimensions (&gt; 100 {micro}m) and high throughputs (10-100 {micro}L/min) necessary to process real-world volumes (1 mL). Previous work demonstrated acoustic focusing of microbeads [4] and biological species [5] in various geometries. We experimentally characterized our device by determining the biological size-cutoff where acoustic radiation pressure forces no longer transport biological particles. Figure 1 shows images of E.Coli ({approx}1 {micro}m) and yeast ({approx}4-5 {micro}m) flowing in a microchannel (200 {micro}m deep, 500 {micro}m wide) at a flow rate of 10 {micro}L/min. The E.Coli does not focus in the acoustic field while the yeast focuses at the channel centerline. This result suggests the acoustic size-cutoff for biological particles in our device lies between 2 and 3 {micro}m. Transverse electrophoresis has been explored extensively in electric field flow fractionation [6] and isoelectric focusing devices [7]. We demonstrated transverse electrophoretic transport of a wide variety of negatively-charged species, including fluorophores, beads, viruses, E.Coli, and yeast. Figure 2 shows the electromigration of a fluorescently labeled RNA virus (MS2) from the lower half of the channel to the upper half region with continuous flow. We demonstrated the effectiveness of our electrophoretically assisted acoustic focusing device by separating virus-like particles (40 nm fluorescent beads, selected to aid in visualization) from a high background concentration of yeast contaminants (see Figure 3). Our device allows for the efficient recovery of virus into a pre-selected purified buffer while background contaminants are acoustically captured and removed. We also tested the device using clinical nasopharyngeal samples, both washes and lavages, and demonstrated removal of unknown particulates (&gt;2 ?m size) from the sample. Our future research direction includes spiking known amounts of bacteria and viruses into clinical samples and performing quantitative off-chip analysis (real-time PCR and flow cytometry).