Purification of bacteriophages using cascade-driven electrokinetic separation

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Bacteriophages, or phages, are viruses with the unique ability to infect and kill bacteria. Many different types of phages exist because each typically targets a specific type of bacteria. This kind of bacterial specificity and lethality has great potential as a means to control antibiotic-resistant, pathogenic bacteria, which cause an estimated annual 2.8 million infections and 35,000 deaths in the United States alone. Before a biomanufactured phage can be used as a treatment by doctors or healthcare professionals, however, it must be purified to remove any remaining pathogenic cells or cell debris that could make a patient very ill. This purification step remains a significant hurdle limiting the use of phage therapy, as no single purification method is suitable for all phage types. In fact, many phages do not survive the most common purification procedures. Thus, there is an urgent need to develop a widely applicable phage purification strategy such that their potential therapeutic value can be realized. One possible solution is to apply electric fields within microfluidic systems to manipulate phages inside the microchannels via electrokinetic effects. This project will examine the efficacy of electrokinetic-based separation methods for rapidly purifying viable phages. The research outcomes are expected to advance the current state-of-the-art in phage purification as well as inform the broader field of electrokinetics. Access to purified phages is essential to the development of novel phage therapies, which are a growing alternative treatment for antibiotic-resistant bacterial infections. Accordingly, this project aims to develop a novel phage purification workflow based on electrokinetics that ensures both viability and purity. The team will apply a technique called insulator-based electrokinetics, wherein microfluidic channels that contain embedded insulating structures are employed. When an electric potential is applied to the channel, the insulating structures distort the electric field distribution inside the channel, creating zones where the electric field is more intense. The strong electrokinetic effects that arise in these zones will enable the rapid and selective concentration of the target phages based upon the unique electrokinetic signature displayed by each phage. A two-stage process to purify the phages will be employed. The first stage will remove the host cells and cell debris, and the second stage will selectively concentrate the phages of interest while allowing other similar particles to pass and be removed. Preliminary experiments demonstrate that phage viability can be preserved in these purification devices. The research objectives are to (1) define the electrokinetic signatures of a diverse set of phages; (2) formulate purification protocols that optimize phage stability during the electrokinetic separation of virions from cellular material; and (3) characterize the molecular composition of the electrokinetics-treated phage samples. The project is a collaborative endeavor that weaves together concepts from engineering and biological sciences and will, thus, provide unique interdisciplinary research opportunities for undergraduate and graduate students. The Rochester Institute of Technology’s Women in Engineering and Women in Science programs will be leveraged to broaden the participation of women in STEM activities.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.