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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI096226-03
Application #
8504898
Study Section
Special Emphasis Panel (ZAI1-BLG-M (M2))
Program Officer
Rao, Malla R
Project Start
2011-08-15
Project End
2016-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
3
Fiscal Year
2013
Total Cost
$1,105,516
Indirect Cost
$207,489
Name
California Institute of Technology
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
Lee, Seung Ah; Yang, Changhuei (2014) A smartphone-based chip-scale microscope using ambient illumination. Lab Chip 14:3056-63
Jung, Jae Hee; Han, Chao; Lee, Seung Ah et al. (2014) Microfluidic-integrated laser-controlled microactuators with on-chip microscopy imaging functionality. Lab Chip 14:3781-9
Kim, Jinho; Erath, Jessey; Rodriguez, Ana et al. (2014) A high-efficiency microfluidic device for size-selective trapping and sorting. Lab Chip 14:2480-90
Han, Chao; Yang, Changhuei (2014) Viral plaque analysis on a wide field-of-view, time-lapse, on-chip imaging platform. Analyst 139:3727-34
Lee, Seung Ah; Erath, Jessey; Zheng, Guoan et al. (2014) Imaging and identification of waterborne parasites using a chip-scale microscope. PLoS One 9:e89712
Han, Chao; Pang, Shuo; Bower, Danielle V et al. (2013) Wide field-of-view on-chip Talbot fluorescence microscopy for longitudinal cell culture monitoring from within the incubator. Anal Chem 85:2356-60
Ah Lee, Seung; Ou, Xiaoze; Lee, J Eugene et al. (2013) Chip-scale fluorescence microscope based on a silo-filter complementary metal-oxide semiconductor image sensor. Opt Lett 38:1817-9
Zheng, Guoan; Ou, Xiaoze; Horstmeyer, Roarke et al. (2013) Characterization of spatially varying aberrations for wide field-of-view microscopy. Opt Express 21:15131-43
Pang, Shuo; Han, Chao; Erath, Jessey et al. (2013) Wide field-of-view Talbot grid-based microscopy for multicolor fluorescence imaging. Opt Express 21:14555-65
Lee, Seung Ah; Zheng, Guoan; Mukherjee, Nandini et al. (2012) On-chip continuous monitoring of motile microorganisms on an ePetri platform. Lab Chip 12:2385-90