EAGER: Addressing the Cyclospora Cayetanensis Detection Gap: A DNA Aptamer and Microfluidic Device Approach

感染水的细菌和寄生虫最终往往会附着在农产品上，威胁人类健康。其中，一些寄生虫，如卡耶坦环孢子虫，很难或不可能培养，这往往造成了难以克服的障碍，阻碍了对它们的有效检测。由于缺乏专门和容易获得的诊断工具来防止受感染的农产品最终出现在人们的餐桌上，往往导致疾病爆发。该项目通过开发一种名为DNA适体的新分子来解决这一关键空白，这种新分子可以与cayetanensis环孢子虫寄生虫特异性结合，然后利用它们来制造一种用户友好的纸质测试。这项工作的目的是能够快速、负担得起和准确地检测这种寄生虫，特别是在不易获得专门实验室设施的地区。该项目通过发现cayetancyclosopora寄生虫的首个DNA适体来解决科学认识问题，并具有通过预防疾病爆发显着改善公众健康的潜力。该项目的目标是发现第一个能够识别卡耶坦环孢子虫的DNA适体，并将其整合到基于纸张的微流体设备中，用于现场诊断。由于我们对这种独特的病原体缺乏了解，以前为实现这一目标所做的大多数努力都受到了限制。与其他球虫寄生虫不同，如刚地弓形虫（弓形虫已被广泛研究，宿主范围更广），卡耶坦环孢子虫是单源球虫，只感染人类肠细胞。这种宿主特异性，加上缺乏关于其膜结构和感染机制的全面数据，使其成为一个异常困难的靶标。因此，该项目的方法与EAGER资助机制一致，解决了对一种特异性结合cayetancyclospora的生物识别元件的未满足需求，这是开发有效检测水源中这种不可培养的单株球虫的定点诊断方法的必要步骤。该项目主要包括两个目标：(1)通过指数富集配体的系统进化（SELEX）过程设计和选择高选择性和稳定的环孢虫适体；(2)开发和测试基于纸张的微流控装置（μPAD）和颜色分析系统，用于检测环孢虫特异性抗原。这些努力将有助于深入了解不可培养病原体的检测，并为水源中的这种寄生虫提供第一个快速、低成本和高度可及的诊断工具。该项目的社会影响在于，它有可能成为开发针对其他具有挑战性病原体的类似诊断工具的典范。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。

Bacteria and parasites that infect water often end up on produce, which threatens human health. Among these, some parasites, such as Cyclospora cayetanensis, are very difficult or impossible to culture, which creates often insurmountable barriers standing in the way of efforts of their effective detection. The lack of specific and easily accessible diagnostic tools that could prevent infected produce ending up on people’s tables often leads to outbreaks of disease. The project addresses this critical gap by developing new molecules, named DNA aptamers, that bind specifically to the Cyclospora cayetanensis parasite and then using them to fabricate a user-friendly paper-based test. This work aims to enable rapid, affordable, and accurate detection of this parasite, especially in areas where specialized laboratory facilities are not easily available. The project addresses both scientific understanding by discovering the first DNA aptamer for the Cyclsopora cayetanensis parasite and holds the potential to significantly improve public health by preventing disease outbreaks.This project's goal is to discover the first DNA aptamers that specifically recognize Cyclospora cayetanensis and integrate them into a paper-based microfluidic device for field-ready diagnostics. Most previous efforts towards meeting this goal have been limited by our lack of through understanding of this unique pathogen. Unlike other coccidian parasites, such as Toxoplasma gondii, which has been extensively studied and has a broader range of hosts, Cyclospora cayetanensis is a monoxenous coccidian, infecting the enterocytes of humans exclusively. This host specificity, paired with the lack of comprehensive data on its membrane structure and infection mechanisms, makes it an exceptionally difficult target. Thus, this project’s approach, aligned with the EAGER funding mechanism, addresses the unmet need for a biological recognition element that is specifically binding to Cyclospora cayetanensis, which is a necessary step for the development of point-of-need diagnostics effective for the detection of this unculturable monoexenous coccidian in water sources. The project involves two main objectives: (1) design and select highly selective and stable Cyclsopora cayetanensis aptamers through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process and (2) develop and test a paper-based microfluidic device (μPAD) coupled with a color analysis system for detecting specific Cyclospora cayetanensis antigens. These efforts will contribute to an in-depth understanding of unculturable pathogen detection and provide a the first rapid, low-cost, and highly accessible diagnostic tool for this parasite in water sources. The societal impact of this project lies in its potential to serve as a model for developing similar diagnostic tools for other challenging pathogens.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.