Multiplex fluorescence optofluidic microscopy for diagnosis of enteric parasites

描述（由申请人提供）：该申请旨在通过一个由加州理工学院生物光子学小组、纽约大学两个寄生虫学小组和Aptina Imaging（传感器芯片的领先创新者和制造商）组成的合作团队，实现一种检测生物防御肠道寄生虫的综合方法。具体来说，我们的目标是检测内阿米巴原虫、贾第鞭毛虫、隐孢子虫和环孢子虫等寄生虫，这些寄生虫通过饮用水污染对公众健康构成威胁。目前对所有这四种寄生虫的诊断是通过对染色的粪便样本进行显微镜检查，其中一些可以通过抗原检测试验得到证实。我们建议实现一个芯片级高分辨率光流体显微镜（OFM）系统，该系统能够以简化和经济有效的方式检测和分析这些寄生虫的彩色和荧光成像。使用抗原特异性OFM荧光检测对囊肿进行自动显微检测，不仅允许对四种不同的病原体进行多重、单步检测，而且还提供了自动诊断，从而消除了对临床训练人员的需求。它还将扩大吞吐率，减少诊断所需的时间和人力。我们还将整合磁性样品浓缩技术，以消除在现场分析中需要基于离心机的浓缩。在试剂方面，我们建议开发用于直接免疫荧光的单克隆抗体，该抗体将针对整个囊肿或定义的囊肿特异性抗原的重组形式产生。拟议中的项目将在两个主要方面有利于生物防御。首先，拟议的OFM系统和相关的抗体实施将直接受益于当前的临床实践，通过简单，即用即放，低成本和自动化的成像细胞术分析取代载玻片制备和显微镜成像步骤。其次，增加磁性样品浓度将消除对离心机的需求，并创建一套广泛可用且非常适合处理紧急情况的技术。

DESCRIPTION (provided by applicant): This application aims to implement a comprehensive approach for the detection of biodefense enteric parasites through a collaborative team comprising of the Caltech biophotonics group, two NYU parasitology groups and Aptina Imaging - a leading innovator and maker of sensor chips. Specifically, we aim to detect Entamoeba, Giardia, Cryptosporidium and Cyclospora, parasites, which pose a public health risk via drinking water supply contamination. Diagnosis of all four of these parasites is currently performed by microscopy of stained stool samples, some of which can be confirmed by antigen-detection tests. We propose to implement a chip-scale high-resolution optofluidic microscopy (OFM) system that is capable of color and fluorescence imaging to detect and analyze these parasites in a streamlined and cost- effective manner. Automated microscopic detection of cysts using antigen specific OFM fluorescence detection will not only allow a multiplex, single-step approach for four different pathogens, but will also provide an automated diagnosis, eliminating the need for clinically-trained personnel. It will also scale up the throughput rate and reduce the time and labor required for diagnosis. We will also integrate magnetic sample concentration technology to eliminate the need for centrifuge-based concentration in field analysis. On the reagent front, we propose to develop monoclonal antibodies for direct immunofluorescence that will be generated against whole cysts or recombinant forms of defined cyst-specific antigens. The proposed project will benefit biodefense in two major ways. First, the proposed OFM system and associated antibody implementation will directly benefit current clinical practices by replacing the slide prep and microscopy imaging step with a simple, drop-and-go, low cost and automatable imaging cytometry analysis. Second, the addition of magnetic sample concentration will eliminate the need for a centrifuge and create a technology set that is broadly usable and well suited to address emergency scenarios. The specific aims of the proposal are as follows: 1. Implement a color and fluorescence OFM system. Able to image at rate of 200 particles/min. 2. Generate monoclonal antibodies that specifically recognize cyst antigens for OFM detection. 3. Develop an algorithm to identify parasites and standardization of the assay. The algorithm will screen the collected images and select the relevant ones, providing images and an automated diagnosis. 4. Scale up number of OFM system per chip to boost total system throughput rate. Develop and implement 10 OFM systems on a single chip to achieve a throughput rate per chip of 2000 particles/min. Aptina Imaging will implement a foundry run to create a chip that contains 50 OFM systems per chip. 5. Develop magnetic antibody tagging separation to directly concentrate parasites from stool samples. We aim to develop a simple sample concentration procedure that eliminates the need for a centrifuge.