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

感染水的细菌和寄生虫往往最终出现在农产品上，这威胁到人类健康。其中，一些寄生虫，如卡氏环孢子虫，很难或不可能培养，这往往会造成难以逾越的障碍，阻碍了有效检测它们的努力。缺乏可以防止受感染产品最终出现在人们餐桌上的具体和容易获得的诊断工具，往往会导致疾病暴发。该项目通过开发名为DNA适配子的新分子来解决这一关键缺口，该分子专门与卡耶坦环孢菌寄生虫结合，然后使用它们来制作一种用户友好的纸质测试。这项工作旨在实现对这种寄生虫的快速、负担得起和准确的检测，特别是在不易获得专门实验室设施的地区。该项目通过发现第一个针对卡氏环孢子虫寄生虫的DNA适配子来解决科学问题，并有可能通过预防疾病暴发来显著改善公众健康。该项目的目标是发现第一个专门识别卡耶坦环孢菌的DNA适配子，并将它们集成到纸基微流控设备中进行现场诊断。以前为实现这一目标所做的大多数努力都受到我们对这一独特病原体缺乏深入了解的限制。与其他球虫不同，如弓形虫，它被广泛研究，宿主范围更广，卡氏环孢子虫是一种单孢球虫，只感染人类的肠道细胞。这种宿主特异性，再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，该项目的办法与急切的供资机制相一致，解决了对特定结合卡耶坦环孢子虫的生物识别元件的未得到满足的需求，这是开发有效检测水源中这种不可培养的单性球虫的需求点诊断方法的必要步骤。该项目涉及两个主要目标：(1)通过指数富集型配体的系统进化(SELEX)过程设计和选择高选择性和稳定的环孢子虫适配子；(2)开发和测试纸基微流控装置(μPAD)和颜色分析系统，用于检测卡氏环孢子虫的特异性抗原。这些努力将有助于深入了解不可培养的病原体检测，并为水源中这种寄生虫提供第一个快速、低成本和高度可获得的诊断工具。该项目的社会影响在于它有可能成为为其他具有挑战性的病原体开发类似诊断工具的典范。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.