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: 8504898
• 负责人: CHANGHUEI YANG
• 金额: $ 110.55万
• 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/8504898
• 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.

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传感器芯片领先创新者和制造商 Aptina Imaging 组成的协作团队，实施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、贾第虫、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测试来确认。我们建议实施一种芯片级高分辨率光流控显微镜（OFM）系统，该系统能够进行彩色和荧光成像，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、单步方法，而且还可以提供自动诊断，从而无需经过临床培训的人员。它还将提高吞吐率并减少诊断所需的时间和劳动力。我们还将集成磁性样品浓缩技术，以消除现场分析中基于离心机浓缩的需要。在试剂方面，我们建议开发用于直接免疫荧光的单克隆抗体，这些抗体将针对整个包囊或特定包囊特异性抗原的重组形式产生。拟议的项目将从两个主要方面有利于生物防御。首先，所提出的 OFM 系统和相关抗体实施将通过用简单、即插即用、低成本和自动化的成像细胞计数分析取代载玻片准备和显微镜成像步骤，直接有益于当前的临床实践。其次，增加磁性样品浓缩功能将消除对离心机的需求，并创建一套广泛使用且非常适合解决紧急情况的技术。 该提案的具体目标如下： 1. 实施颜色和荧光 OFM 系统。能够以 200 个粒子/分钟的速度成像。 2. 生成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标准化测定。该算法将筛选收集到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。在单芯片上开发并实现 10 个 OFM 系统，实现每芯片 2000 个粒子/分钟的吞吐率。 Aptina Imaging 将实施代工生产，以创建每个芯片包含 50 个 OFM 系统的芯片。 5. 开发磁性抗体标记分离技术，直接浓缩粪便样本中的寄生虫。我们的目标是开发一种简单的样品浓缩程序，无需离心机。