Abstract
Handling of submicron-sized objects is important in many biochemical and biomedical applications, but few methods today can precisely manipulate this range of particles. We present gradient acoustic focusing that enables flow-through particle separation of submicron particles and cells and we apply it for separation of bacteria from blood lysate to facilitate their detection in whole blood for improved diagnostics. To control suspended objects below the classical 2µm size limit for acoustic focusing, we introduce a co-flowing acoustic impedance gradient to generate a stabilizing acoustic volume force that supresses acoustic streaming. The method is validated theoretically and experimentally using polystyrene particles, Staphylococcus aureus, Streptococcus pneumoniae and Escherichia coli. The applicability of the method is demonstrated by the separation of bacteria from selectively chemically lysed blood. Combined with downstream operations, this new approach opens up for novel methods for sepsis diagnostics.
Highlights
Microfluidic flow-through channel networks can be employed to separate or analyze biological particles by their fluorescent[1], optical[2], electrical[3,4,5], magnetic[6,7] or bio-mechanical properties[8,9,10,11,12]
Purification of small particles, e.g. bacteria and platelets[25,26,27,28] has been achieved by acoustically pushing away the larger particles from the smaller particles, but this approach does not enable isolation of sub-micron-sized biological particles from a complex background of molecules, particles or debris that are of smaller size, which is essential for purification protocols
For bulk acoustic waves, acoustic streaming can be greatly reduced by introducing a gradient in acoustic impedance in the acoustic cavity by standard gradient centrifugation media[32,33]
Summary
Microfluidic flow-through channel networks can be employed to separate or analyze biological particles by their fluorescent[1], optical[2], electrical[3,4,5], magnetic[6,7] or bio-mechanical properties[8,9,10,11,12]. Efficient suppression of streaming opens up for acoustic separation of particles below the classical size limit and thereby enables transfer of micrometer- and sub-micrometer-sized particles in continuous flow from one medium to another using acoustic radiation forces, inspiring new applications of bulk acoustic waves involving platelets, exosomes, large organelles and bacteria. Several methods are available for direct nucleic acid extraction and detection using polymerase chain reaction (PCR), none has yet been able to replace blood culture[37] One reason for this is the low number of bacteria (frequently less than 200 colony forming units per milliliter blood) in human sepsis[38]. The lysis-centrifugation method is not in routine clinical use, due to contamination risks and labor intensive handling[42], which are hurdles that can be diminished by chip-integrated microfluidic approaches for blood cell lysis[43,44] and sample concentration[26,45]
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