Purification of bacteriophages using cascade-driven electrokinetic separation

噬菌体是一种具有感染和杀死细菌的独特能力的病毒。存在许多不同类型的细菌，因为每种细菌通常针对特定类型的细菌。这种细菌的特异性和致命性具有很大的潜力，可以作为控制耐药性致病细菌的手段，仅在美国，这种细菌每年就造成280万例感染和35，000例死亡。然而，在生物制造的噬菌体可以被医生或医疗保健专业人员用作治疗之前，它必须被纯化以去除任何剩余的致病细胞或细胞碎片，这些细胞或细胞碎片可能会使患者病情严重。该纯化步骤仍然是限制噬菌体治疗使用的重要障碍，因为没有单一的纯化方法适用于所有噬菌体类型。事实上，许多细菌在最常见的净化程序中无法存活。因此，迫切需要开发一种广泛适用的噬菌体纯化策略，以实现其潜在的治疗价值。一种可能的解决方案是在微流体系统内施加电场，以通过电动效应操纵微通道内的微流体。本项目将研究基于电动分离方法的有效性，用于快速纯化活性微生物。该研究成果有望推动噬菌体纯化的当前最新技术水平，并为更广泛的电动力学领域提供信息。获得纯化的噬菌体对开发新型噬菌体疗法至关重要，这是一种日益增长的耐药细菌感染的替代治疗方法。因此，该项目旨在开发一种基于电动力学的新型噬菌体纯化工作流程，以确保活力和纯度。该团队将应用一种称为基于绝缘体的电动力学的技术，其中使用包含嵌入式绝缘结构的微流体通道。当向通道施加电势时，绝缘结构扭曲通道内的电场分布，产生电场更强的区域。在这些区域中出现的强动电效应将使得能够基于由每个噬菌体展示的独特动电特征来快速和选择性地浓缩靶向药物。将采用两个阶段的工艺来纯化水。第一阶段将去除宿主细胞和细胞碎片，第二阶段将选择性地浓缩感兴趣的颗粒，同时允许其他类似的颗粒通过并被去除。初步实验表明，噬菌体活力可以在这些纯化装置中得到保留。研究目标是：（1）定义一组不同的噬菌体的电动签名;（2）制定纯化方案，优化从细胞材料中电动分离病毒粒子的过程中的噬菌体稳定性;和（3）表征电动处理的噬菌体样品的分子组成。该项目是一项合作奋进，将工程和生物科学的概念编织在一起，从而为本科生和研究生提供独特的跨学科研究机会。罗切斯特理工学院的女性工程和女性科学项目将被用来扩大女性在STEM活动中的参与。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.