Multiplex fluorescence optofluidic microscopy for diagnosis of enteric parasites

描述(申请人提供)：本申请旨在通过一个由加州理工学院生物光子学小组、两个纽约大学寄生虫学小组和领先的传感器芯片创新者和制造商Aptina成像公司组成的合作团队，实施一种全面的生物防御性肠道寄生虫检测方法。具体地说，我们的目标是检测内阿米巴、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供应受到污染，构成公共健康风险。目前，对所有四种寄生虫的诊断都是通过对染色的粪便样本进行显微镜检查来进行的，其中一些可以通过抗原检测测试来确认。我们建议实现一个芯片规模的高分辨率光学流体显微镜(OFM)系统，该系统能够进行彩色和荧光成像，以简化和经济高效的方式检测和分析这些寄生虫。使用抗原特异性荧光检测对包囊进行自动显微检测不仅可以对四种不同的病原体进行多重、一步法检测，而且还可以提供自动诊断，而不需要经过临床培训的人员。它还将提高吞吐量，并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，以消除现场分析中使用离心机浓缩的需要。在试剂方面，我们建议开发用于直接免疫荧光的单抗，这种抗体将针对整个包囊或重组形式的已定义包囊特异性抗原产生。拟议中的项目将在两个主要方面使生物防御受益。首先，拟议的OFM系统和相关抗体的实施将直接受益于当前的临床实践，它将用一种简单、即插即用、低成本和自动化的成像细胞术分析取代载玻片准备和显微镜成像步骤。其次，磁性样品浓缩的增加将消除对离心机的需求，并创造出一套可广泛使用、非常适合应对紧急情况的技术。 该建议的具体目标如下： 1.实现了彩色和荧光OFM系统。能够以每分钟200个粒子的速度成像。 2.制备特异性识别包囊抗原的单抗，用于OFM检测。 3.建立识别寄生虫的算法和标准化检测方法。该算法将对收集的图像进行筛选并选择相关图像，提供图像和自动诊断。 4.扩展每个芯片的OFM系统数量，提高系统总吞吐能力。在一个芯片上开发和实施10个OFM系统，以实现每芯片2000个粒子/分钟的吞吐速率。阿普蒂纳成像将实施代工运行，以创建一个芯片，每个芯片包含50个OFM系统。 5.建立磁性抗体标记分离技术，直接从粪便标本中浓缩寄生虫。我们的目标是开发一种简单的样品浓缩程序，消除对离心机的需求。

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.