Grantee Research Project Results
Final Report: A Hybrid Pathogen Detection System
EPA Contract Number: EPD06088Title: A Hybrid Pathogen Detection System
Investigators: Aguilar, Zoraida P.
Small Business: Vegrandis Inc.
EPA Contact: Richards, April
Phase: II
Project Period: April 1, 2006 through June 30, 2007
Project Amount: $225,000
RFA: Small Business Innovation Research (SBIR) - Phase II (2006) Recipients Lists
Research Category: Drinking Water , SBIR - Water and Wastewater , Small Business Innovation Research (SBIR)
Description:
This project is focused on the development of disposable self-contained microelectrochemical microarray hybrid cartridge (SMEHA) for the quantification and viability assessments of waterborne pathogens to meet the escalating needs for fast warnings during pathogen outbreaks and the possible dispersal of biological disposable warfare agents. The SMEHA cartridge can be customized for any pathogen of interest and we had chosen to show its applications for the detection of C. parvum and G. lamblia, which are two of the most resilient waterborne pathogens that are found not only in the United States but worldwide. Both of these protozoans seriously threaten the nation’s water supply because they resist ordinary water treatment processes and do not respond to common antibiotics. Because of the speed, the specificity, and ability to differentiate between dead and live organisms using our SMEHA system, investigators will be able to quickly act on suspected Cryptosporidium or Giardia contamination. Both the flow and the path of contamination can be quickly and efficiently traced. The development of the disposable SMEHA cartridge, the reagent kits, and the fully automated instrument will advance the EPA Method 1622 and 1623 by providing a previously unavailable combination of speed, sensitivity, affordability, ease-of-use, and test for viability of waterborne pathogens.
Summary/Accomplishments (Outputs/Outcomes):
We have developed:
- three kinds of disposable self-contained microelectrochemical microarray hybrid cartridges, which are dedicated for:
1) the detection of C. parvum oocysts only
2) the detection of Giardia cysts only
3) the detection of both C. parvum oocysts and Giardia cysts
- three kinds of reagent kits containing all the solutions necessary to perform the capture and test for viability using the disposable cartridge for:
1) the detection of C. parvum oocysts only
2) the detection of Giardia cysts only
3) the detection of both C. parvum oocysts and Giardia cysts
- the operations manual for the instrument and protocol manual for assays using the disposable self-contained microelectrochemical hybrid assay (SMEHA) cartridge that includes standard operating procedures, trouble shooting guidelines, instrument and assay checklist, data forms, and laboratory processing forms for documentation.
The chip based self-contained micro-electrochemical array assay detection of Cryptosporidium parvum oocysts and Giardia lamblia uses 50-μm diameter cavities on a chip. The cavities have built-in microelectrodes that allows performance of electrochemical detection of waterborne pathogens without the need for introducing external electrodes. The two-fold specificity of this analysis arising from the IgG1 (which shows reduced binding to algae debris and minerals in environmental samples thereby increasing sensitivity of detection) capture and the release of hsp70 mRNA eliminates the false positives and false negatives for Cryptosporidium parvum.
The automated immunoassay portion of the SMEHA DC was demonstrated for the detection of as low as 1 oocyst/mL C. parvum and G. lamblia in a less than 30 minute total assay time from capture to signal generation. The method developed in this project can be applied following the EPA Method 1623 where 10 L of water samples are filtered and the collected residue on the filter is backwashed with the clean filtered water to reconstitute all the solids from the filter. Instead of applying IFA to the reconstituted pellet followed by microscopy, the pellet will be passed through the DC and the sandwich ELISA with electrochemical detection will be used to initially verify the presence of the organisms. This is followed by heat shocking that will be a built-in feature of the instrument (it will be incorporated after all the solution delivery components have been finalized) through micro-heaters. Cavities adjacent to the immunoassay capture sites are used for hsp mRNA capture that are used to verify viability of the captured organisms. Automated detection of 100 cysts of G. lamblia in the presence of 100 oocysts of C. parvum was exhibited using the SMEHA cartridge. Furthermore, the viability of the organisms were tested and exhibited through the production and detection of hsp mRNA in the individual assays and also in the multiplex assay.
The shelf life of the primary antibody functionalized chip was established at 30 days when kept in the refrigerator between 0 to 10ºC. The secondary antibody in solution also can be kept for 30 days in the refrigerator between 0 to 10ºC. The buffers and other components of the reagent kit for the immunoassay capture of both C. parvum and G. lamblia showed a shelf life of at least 150 days, which was the time in which we had to perform the studies. We will continue with these studies even after this project has ended in order to determine the maximum period of time that the reagents can be stored without loss in potency.
Conclusions:
The disposable SMEHA cartridge was developed for the automated detection of waterborne pathogens in a 30-minute total assay time from capture to signal generation. Both C. parvum and G. lamblia were detected at 1 oocyst/mL sample solution. The viability of both organisms was exhibited through the detection of hsp mRNA that was released after a 20-minute heating at 42 to 45ºC. The released hsp mRNA were detected in microcavities modified with the capture probes.
The NSA signals in gold during the immunoassay and DNA-hybridization assays were eliminated using proprietary blocking buffers. The NSA in the various components of the DC and the components of the automated delivery of sample and reagents (channel material, luers and ferrules, and capillary tubing) also were minimized or completely eliminated by soaking in specific blocking buffers.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 1 publications | 1 publications in selected types | All 1 journal articles |
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Type | Citation | ||
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Aguilar ZP, Van Nguyen C, Sirisena M, Gertsch J, Arumugam P, Spencer D, Wansapura C, Aguilar Y, Homesley J. Automated microarray technology for biomedical and environmental sensors. ECS Transactions 2006;3(10):125-137. |
EPD06088 (Final) |
not available |
Supplemental Keywords:
small business, SBIR, EPA, drinking water, water contamination, self-contained microelectrochemical hybrid assay, SMEHA, reagent kits, waterborne protozoan pathogens, Giardia, Cryptosporidium, ecosystem protection/environmental exposure & risk, water, scientific discipline, RFA, physical aspects, drinking water, health risk assessment, physical processes, environmental chemistry, monitoring/modeling, drinking water system, drinking water contaminants, public health, water quality, exposure and effects, safe drinking water, water quality parameters, drinking water distribution system, mRNA hybridization assay, cryptosporidium parvum oocysts, monitoring, immunofluorescent assay, fecal contamination, pathogens, waterborne disease, microelectrochemical detection, exposure, microbial contamination, microbial risk assessment, biosensors, bioanalytical devices, waterborne pathogen, electrochemical instrument,, RFA, Scientific Discipline, PHYSICAL ASPECTS, Water, Ecosystem Protection/Environmental Exposure & Risk, Environmental Chemistry, Health Risk Assessment, Monitoring/Modeling, Physical Processes, Drinking Water, cryptosporidium parvum oocysts, microbial contamination, Safe Drinking Water, pathogens, monitoring, microbial risk assessment, assays, water quality parameters, waterborne disease, exposure and effects, fecal contamination, mRNA hybridization assay, exposure, drinking water distribution system, cryptosporidium , public health, water quality, drinking water contaminants, immunofluorescent assay, drinking water systemSBIR Phase I:
A Hybrid Pathogen Detection System | Final ReportThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.