Phages used for phage therapy of multidrug resistant bacteria must be highly purified prior to use. There are limited purification approaches that are broadly applicable to many phage types. Electrokinetics has shown great potential to manipulate phages, but obstructions from the cell debris produced during phage propagation can severely diminish the capacity of an electrokinetic device to concentrate and purify phage samples. A multipart insulator‐based electrokinetic device is proposed here to remove the larger, undesirable components of mixtures from phage preparations while transferring the freshly purified and concentrated sample to a second stage for downstream analysis. By combining the large debris prescreen and analysis stages in a streamlined system, this approach simultaneously reduces the impact of clogging and minimizes the sample loss observed during manual transferring of purified samples. Polystyrene particles were used to demonstrate a diminished sample loss of approximately one order of magnitude when using the cascade device as opposed to a manual transfer scheme. The purification and concentration of three different phage samples were demonstrated using the first stage of the cascade device as a prescreen. This design provides a simple method of purifying and concentrating valuable samples from a complex mixture that might impede separation capacity in a single channel.
This content will become publicly available on March 1, 2025
Bacteriophage therapy presents a promising avenue for combating antibiotic-resistant bacterial infections. Yet, challenges exist, particularly, the lack of a straightforward purification pipeline suitable for widespread application to many phage types, as some phages are known to undergo significant titer loss when purified via current techniques. Electrokinetic methods offer a potential solution to this hurdle, with nonlinear electrophoresis emerging as a particularly appealing approach due to its ability to discern both the size and shape of the target phage particles. Presented herein is the electrokinetic characterization of the mobility of nonlinear electrophoresis for two phages (SPN3US and ϕKZ) and three types of polystyrene nanoparticles. The latter served as controls and were selected based on their sizes and surface charge magnitude. Particle tracking velocimetry experiments were conducted to characterize the mobility of all five particles included in this study. The results indicated that the selected nanoparticles effectively replicate the migration behavior of the two phages under electric fields. Further, it was found that there is a significant difference in the nonlinear electrophoretic response of phages and that of host cells, as first characterized in a previous report, illustrating that electrokinetic-based separations are feasible. The findings from this work are the first characterization of the behavior of phages under nonlinear electrophoresis effects and illustrate the potential for the development of electrokinetic-based phage purification techniques that could aid the advancement of bacteriophage therapy.
more » « less- Award ID(s):
- 2133207
- PAR ID:
- 10528781
- Publisher / Repository:
- Multidisciplinary Digital Publishing Institute
- Date Published:
- Journal Name:
- Micromachines
- Volume:
- 15
- Issue:
- 3
- ISSN:
- 2072-666X
- Page Range / eLocation ID:
- 369
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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