Multiplex fluorescence optofluidic microscopy for diagnosis of enteric parasites

多重荧光光流控显微镜诊断肠道寄生虫

• 批准号: 8186373
• 负责人: CHANGHUEI YANG
• 金额: $ 120.89万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-15 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8186373
• 关键词: Address; Algorithms; Animals; Antibodies; Antigens; Aptina; Biological; Biological Assay; Biophotonics; Clinical Sensitivity; Color; Coupled; Cryptosporidium; Cryptosporidium parvum; Cyclospora; Cyst; Detection; Diagnosis; Diagnostic Procedure; Direct immunofluorescence; Disease Outbreaks; Disinfection; Drops; Dyes; Emergency Situation; Engineering; Entamoeba; Entamoeba histolytica; Enteral; Equipment; Feces; Fluorescence; Fluorescence Microscopy; Formalin; Giardia; Giardia lamblia; Gold; Human; Human Resources; Image; Image Cytometry; Immunomagnetic Separation; Infection; Magnetism; Microfluidics; Microscope; Microscopic; Microscopy; Monoclonal Antibodies; Oocysts; Organism; Parasites; Parasitology; Phase; Procedures; Process; Protozoa; Public Health; Quantum Dots; Reagent; Recombinants; Reproducibility; Research; Resistance; Resolution; Risk; Running; Sampling; Sensitivity and Specificity; Signal Transduction; Slide; Specificity; Specimen; Staining method; Stains; Standardization; System; Techniques; Technology; Testing; Time; Training; Water; Water Supply; Work; Yang; base; biodefense; cellular imaging; clinical practice; cost; cost effective; design; disease diagnosis; drinking water; fluorescence imaging; particle; pathogen; scale up; sensor

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.

描述（由申请人提供）：本申请旨在通过一个由Caltech Biophotonics小组组成的协作团队，两个NYU寄生虫学组和Aptina Imaging（Aptina Imaging）实施全面的方法来检测生物forense肠寄生虫 - 一个领先的创新者和制造商传感器芯片。具体而言，我们旨在检测Entamoeba，Giardia，隐孢子虫和环孢子虫，寄生虫，它们通过饮用水供应污染构成公共卫生风险。目前，通过染色的粪便样品显微镜进行了所有这些寄生虫的诊断，其中一些可以通过抗原检测测试来确认。我们建议实施芯片尺度的高分辨率光氟化显微镜（OFM）系统，该系统能够进行颜色和荧光成像，以简化且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荧光检测对囊肿的自动显微镜检测不仅将允许四种不同病原体的多重单步方法，而且还将提供自动诊断，从而消除了对临床训练的人员的需求。它还将扩大吞吐量率，并减少诊断所需的时间和人工。我们还将整合磁性样品浓度技术，以消除在现场分析中基于离心机浓度的需求。在试剂方面，我们建议开发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囊肿特异性抗原产生。拟议的项目将以两种主要方式使生物陷入困境。首先，提出的OFM系统和相关抗体实施将通过简单，掉落，低成本和可自动化的成像细胞仪分析代替幻灯片准备和显微镜成像步骤，直接使当前临床实践受益。其次，增加磁性样品浓度将消除对离心机的需求，并创建一个广泛可用且非常适合解决紧急情况的技术集合。 该提案的具体目的如下： 1。实现M系统的颜色和荧光。能够以200个颗粒/分钟的速率图像。 2。产生单克隆抗体，该抗体特异性地识别用于OFM检测的囊肿抗原。 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。每个芯片规模扩大M的系统数量，以提高系统吞吐量。在单个芯片上开发和实现10个OFM系统，以达到2000个颗粒/分钟的每芯片的吞吐率。 Aptina Imaging将实施铸造式运行，以创建一个芯片，该芯片包含每个芯片的50个系统。 5。开发磁抗体标记分离，直接从粪便样品中浓缩寄生虫。我们旨在制定简单的样品浓度程序，以消除对离心机的需求。 公共卫生相关性：我们建议开发芯片尺度和低成本的成像细胞仪以及针对肠寄生虫的单克隆抗体 - Entamoeba，Giardia，Cryptosporidium和Cyclospora。提出的技术将通过简单的“跌落和速度”低成本和可自动化的成像细胞仪分析来代替幻灯片准备和显微镜成像步骤，直接使当前的临床实践受益。此外，我们旨在将免疫磁性分离纳入集中肠寄生虫，以创建不需要重型设备的技术集，广泛可用，非常适合解决紧急情况。