Multiplex fluorescence optofluidic microscopy for diagnosis of enteric parasites

描述（由申请人提供）：本申请旨在通过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Aptina成像（传感器芯片的领先创新者和制造商）组成的协作团队实施用于检测生物防御肠道寄生虫的综合方法。具体而言，我们的目标是检测内阿米巴、贾第虫、隐孢子虫和环孢子虫，这些寄生虫通过饮用水供应污染构成公共卫生风险。目前，所有这四种寄生虫的诊断都是通过染色粪便样本的显微镜检查进行的，其中一些可以通过抗原检测试验来确认。我们建议实现一种芯片级高分辨率光流体显微镜（OFM）系统，该系统能够进行彩色和荧光成像，以流线型和具有成本效益的方式检测和分析这些寄生虫。使用抗原特异性OFM荧光检测的囊肿的自动显微镜检测将不仅允许针对四种不同病原体的多路复用、单步方法，而且还将提供自动诊断，从而消除对经过临床训练的人员的需要。它还将提高吞吐率，减少诊断所需的时间和劳动力。我们还将整合磁性样品浓缩技术，以消除现场分析中对离心浓缩的需要。在试剂方面，我们建议开发用于直接免疫荧光的单克隆抗体，该抗体将针对整个囊肿或重组形式的定义的囊肿特异性抗原产生。拟议的项目将在两个主要方面有利于生物防御。首先，所提出的OFM系统和相关的抗体实施将通过用简单、即插即用、低成本和可自动化的成像细胞术分析代替载玻片制备和显微镜成像步骤而直接有益于当前的临床实践。第二，磁性样品浓缩的增加将消除对离心机的需要，并创造一套广泛适用的技术，非常适合处理紧急情况。 该提案的具体目标如下： 1.实现彩色和荧光OFM系统。能够以200个粒子/分钟的速率成像。 2.产生特异性识别包囊抗原的单克隆抗体用于OFM检测。 3.开发一种算法来识别寄生虫和测定的标准化。该算法将筛选收集的图像并选择相关图像，提供图像和自动诊断。 4.增加每个芯片的OFM系统数量，以提高系统总吞吐率。在单个芯片上开发并实施10个OFM系统，以实现每个芯片2000个粒子/分钟的吞吐率。Aptina Imaging将实施代工运行，以创建每个芯片包含50个OFM系统的芯片。 5.发展磁性抗体标记分离技术，直接从粪便样品中富集寄生虫。我们的目标是开发一种简单的样品浓缩程序，消除对离心机的需要。 公共卫生相关性：我们建议开发一个芯片规模和低成本的成像细胞仪和一套单克隆抗体，针对肠道寄生虫-内阿米巴，贾第虫，隐孢子虫和环孢子虫。所提出的技术将直接受益于当前的临床实践，通过用简单的“即插即用”低成本和可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。此外，我们的目标是将免疫磁性分离作为浓缩肠道寄生虫的一种手段，以创造一种不需要重型设备的技术，该技术广泛适用于解决紧急情况。

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. PUBLIC HEALTH RELEVANCE: We propose to develop a chip-scale and low cost imaging cytometer and a suite of monoclonal antibodies that target the enteric parasites - Entamoeba, Giardia, Cryptosporidium and Cyclospora. The proposed technology 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. Additionally, we aim to incorporate immunomagnetic separation as a means to concentrate enteric parasites to create a technology set that does not require heavy equipment, is broadly usable and well suited to address emergency scenarios.