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噬菌体，因为每种噬菌体通常针对一种特定类型的细菌。这种细菌的特异性和致死率作为一种控制抗生素耐药致病菌的手段具有巨大的潜力。据估计，仅在美国，每年就有280万例感染和3.5万例死亡。然而，在医生或医疗保健专业人员将生物制造的噬菌体用于治疗之前，必须对其进行纯化，以去除任何可能使患者病情严重的残留致病性细胞或细胞碎片。这一纯化步骤仍然是限制噬菌体治疗使用的一个重大障碍，因为没有一种纯化方法适用于所有噬菌体类型。事实上，许多噬菌体在最常见的纯化程序中无法存活。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中施加电场，通过电动效应来操纵微通道内的噬菌体。本项目将研究基于电动分离方法快速纯化活菌体的功效。该研究成果有望推动当前最先进的噬菌体纯化，并为更广泛的电动力学领域提供信息。获得纯化噬菌体对于开发新型噬菌体疗法至关重要，这是治疗抗生素耐药细菌感染的一种日益增长的替代疗法。因此，本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，以确保其活力和纯度。该团队将应用一种称为基于绝缘体的电动力学技术，其中使用包含嵌入式绝缘结构的微流体通道。当电势作用于通道时，绝缘结构扭曲了通道内的电场分布，产生了电场更强的区域。在这些区域产生的强大的电动力效应将使目标噬菌体能够根据每个噬菌体所显示的独特电动力特征快速和选择性地集中。将采用两阶段的过程来纯化噬菌体。第一阶段将清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噬菌体，同时允许其他类似的颗粒通过并被清除。初步实验表明，在这些纯化装置中可以保持噬菌体的活力。研究目标是：(1)定义不同噬菌体的电动力学特征；(2)制定纯化方案，优化病毒粒子与细胞物质电动分离过程中噬菌体的稳定性；(3)表征经电动力学处理的噬菌体样品的分子组成。该项目是一项将工程和生物科学的概念结合在一起的合作努力，因此将为本科生和研究生提供独特的跨学科研究机会。罗彻斯特理工学院的“女性参与工程”和“女性参与科学”项目将被用来扩大女性参与STEM活动的范围。该奖项反映了美国国家科学基金会的法定使命，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.